Methods and treatment of trauma adverse events with oxygen reduced blood

ABSTRACT

Methods for the reversal of hemorrhagic shock or hemorrhagic trauma. Methods for restoring mean arterial pressure to a normal range and reducing trauma adverse risks in a patient through the administration of oxygen reduced blood compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of Ser. No. 16/903,108, filed Jun.16, 2020, which is a continuation of 16/849,614, filed Apr. 15, 2020,which is a continuation of U.S. application Ser. No. 16/791,697, filedFeb. 14, 2020, which is a continuation of U.S. application Ser. No.16/614,683, filed Nov. 18, 2019, which is a U.S. National Stage filingunder 35 U.S.C. § 371 of International Application No.PCT/US2018/033404, filed May 18, 2018, which claims benefit and priorityto U.S. Provisional Application No 62/508,783, filed May 19, 2017, whichis hereby incorporated by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with the United States government support underR44HL132172 awarded by the National Heart, Lung, and Blood Institute.The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to treatment of trauma and hemorrhagicshock.

BACKGROUND OF THE INVENTION

In 2010, there were 5.1 million deaths from injuries, surpassing thenumber of deaths due to HIV, tuberculosis, and malaria combined (3.8million). See Norton, et al., “Global Health Injuries” in The NEAT368:1723-30 (2013) (“Norton 2013”) (hereby incorporated by reference inits entirety). Injuries include unintentional injuries (e.g., road-traffic incidents, falls, and bums) and intentional injuries (e.g.,self-harm, interpersonal violence, war and conflict). See Norton 2013.The number of deaths from injuries increased by 24% between 1990 and2010, worldwide, and increased 23% between 2000 and 2010, in the UnitedStates. See Norton 2013. Additionally, at least 20% of all trauma deathsare the result of survivable injuries and are therefore preventable withoptimal care. Fox et al., “Earlier Endpoints are Required forHemorrhagic Shock Trials Among Severely Injured Patients.” Shock,47:567-73 (2017) (hereby incorporated by reference in its entirety). Thepercentage of preventable deaths make it imperative to develop therapyfor avoidable complications which lead to mortality.

Penetrating wounds (e.g., gunshot or stabbing) and blunt trauma (e.g.,fall or automobile accident) are major causes of hemorrhagic trauma. Theresulting shock is a condition of inadequate oxygen supply to tissuesfrom massive hemorrhage causing oxygen debt, anaerobic metabolism andraise of plasma lactate level. Failure to reverse shock by restoringcirculation and oxygen delivery can result in permanent tissue damage,multiple organ failure and mortality.

Clinical sequelae of hemorrhagic trauma and shock include mortality fromexsanguination within several hours of trauma, as well as after 24 hoursfrom morbidity from trauma and massive transfusion. Such morbidityincludes multiple organ failure including lung, kidney, liver from acutetraumatic coagulopathy or inflammation, and infection/sepsis fromtransfusion related immune modulation; both morbidities are heightenedby lower quality of transfused blood products as well as higher volumeof transfused pRBC.

One approach for treating hemorrhage shock is the use of crystalloidsfor resuscitation. However, the use of crystalloids result in increasedmorbidity and mortality by causing trauma induced coagulopathy. For atleast this reason, early administration of blood components is advocatedfor reversing shock caused by hemorrhagic trauma. Packed red blood cells(pRBCs) are transfused into a hemorrhagic trauma patient to restore lostblood volume, restore oxygen carrying capacity in patients and restoreoxidative metabolism in tissue from anaerobic metabolism. However, theuse of pRBCs is not without risk of complications, including antigenmismatch, pathogen transmission, circulatory overload, and degradationof pRBCs during ex vivo storage.

When stored conventionally, stored blood undergoes a steadydeterioration which is associated with various storage lesionsincluding, among others, hemolysis, hemoglobin degradation, and reducedATP and 2,3-DPG concentrations. When transfused into a patient, theeffects of the steady deterioration during storage manifest, forexample, as a reduction in the 24-hour in vivo recovery. The rapiddecrease in the hematocrit that results from reduced 24-hour recovery,when severe, can result in delayed hemolytic transfusion reaction(DHTR). Other complications, for example systemic inflammatory responsesyndrome (SIRS), transfusion related acute lung injury (TRALI), andtransfusion related immunomodulation (TRIM) are associated withtransfusion of stored blood, though identification of the underlyingcauses has remained unclear.

Even when transfused within the current 6-week limit, stored RBCs tendto exhibit lower quality (e.g. increased fraction of RBCs removed;compromised oxygen exchange capacity; reduced deformability) andincreased toxicity, often manifested as the clinical sequelae oftransfusion therapy. A large and growing number of articles in theliterature supports this view. See Zimring, “Established and theoreticalfactors to consider in assessing the red cell storage lesion,” Blood,125:2185-90 (2015); Zhu et al., “Impaired adenosine-5′-triphosphaterelease from red blood cells promotes their adhesion to endothelialcells: a mechanism of hypoxemia after transfusion,” Critical caremedicine, 39:2478-86 (2011); Weinberg et al., “Red blood cell age andpotentiation of transfusion-related pathology in trauma patients,”Transfusion, 51:867-73 (2011); Spinella et al., “Does the storageduration of blood products affect outcomes in critically ill patients?”Transfusion 51:1644-50 (2011); Roback et al., “Insufficient nitric oxidebioavailability: a hypothesis to explain adverse effects of red bloodcell transfusion,” Transfusion, 51:859-66 (2011); Reynolds et al., “Thetransfusion problem: role of aberrant S-nitrosylation,” Transfusion,51:852-8 (2011); Kim-Shapiro et al., “Storage lesion: role of red bloodcell breakdown,” Transfusion, 51:844-51 (2011); Jy et al.,“Microparticles in stored red blood cells as potential mediators oftransfusion complications,” Transfusion, 51:886-93 (2011); Hod et al.,“Transfusion of human volunteers with older, stored red blood cellsproduces extravascular hemolysis and circulating non-transferrin-boundiron,” Blood, 118:6675-82 (2011); Flegel et al., “Does prolonged storageof red blood cells cause harm?” British journal of haematology 165:3-16(2014); Redlin et al., “Red blood cell storage duration is associatedwith various clinical outcomes in pediatric cardiac surgery,”Transfusion medicine and hemotherapy: offizielles Organ der DeutschenGesellschaft fur Transfusionsmedizin and Immunhamatologie 41:146-51(2014); Rogers et al., “Storage duration of red blood cell transfusionand Clostridium difficile infection: a within person comparison,” PLoSOne 9:e89332 (2014); Spinella et al., “Properties of stored red bloodcells: understanding immune and vascular reactivity,” Transfusion51:894-900 (2011); Brown et al., “Length of red cell unit storage andrisk for delirium after cardiac surgery,” AnesthAnalg, 119:242-50(2014); Wang et al., “Transfusion of older stored blood worsens outcomesin canines depending on the presence and severity of pneumonia,”Transfusion, 54:1712-24 (2014); Liu et al., “Mechanism of faster NOscavenging by older stored red blood cells,” Redox biology, 2:211-9(2014); Prestia et al., “Transfusion of stored blood impairs hostdefenses against Gram-negative pathogens in mice,” Transfusion54:2842-51 (2014); D'Alessandro et al., “An update on red blood cellstorage lesions, as gleaned through biochemistry and omicstechnologies,” Transfusion, 55:205-19 (2015) (hereby incorporated byreference in their entireties). An extensive body of in vitro studiesunequivocally shows the degradation of RBCs (storage lesions) duringconventional storage. A body of emerging metabolomic studies show thedevelopment of storage lesions at the molecular level. See Roback etal., “Metabolomics of AS-1 RBCs Storage,” Transfusion medicine reviews(2014); D'Alessandro et al., “Metabolomics of AS-5 RBCs supernatantsfollowing routine storage,” Vox sanguinis (2014); D'Alessandro et al.,“Routine storage of red blood cell (RBC) units in additive solution-3: acomprehensive investigation of the RBC metabolome,” Transfusion55:1155-68 (2015); D'Alessandro et al., “Red blood cell storage inadditive solution-7 preserves energy and redox metabolism: ametabolomics approach,” Transfusion (2015); Wither et al., “Hemoglobinoxidation at functional amino acid residues during routine storage ofred blood cells,” Transfusion (2015); D'Alessandro et al., “Citratemetabolism in red blood cells stored in additive solution-3,”Transfusion (2016); D'Alessandro et al., “Omics markers of the red cellstorage lesion and metabolic linkage,” Blood Transfus, 15:137-44 (2017)(hereby incorporated by reference in their entireties). There is a needfor reducing or preventing this degradation to increase the efficacy oftransfusions (more O₂ delivery to peripheral tissues immediately aftertransfusion) and to reduce mortality due to hemorrhagic trauma.

Oxidative damage initiates many RBC storage lesions in conventionallystored blood and their downstream consequences; thus, methods to reducethe extent of oxidative stress are required to reduce the RBC storagelesions. A number of approaches have been developed aimed at minimizingstorage lesions and improving transfusion outcomes. Approaches includeadditive solutions (for example, U.S. Pat. No. 4,769,318 to Hamasaki etal. and U.S. Pat. No. 4,880,786 to Sasakawa et al. U.S. Pat. No.6,447,987 to Hess et al.), frozen storage (see U.S. Pat. No. 6,413,713to Serebrennikov Chaplin et al., “Blood Cells for Transfusion,” Blood,59: 1118-20 (1982), and Valeri et al., “The survival, function, andhemolysis of human RBCs stored at 4 degrees C. in additive solution(AS-1, AS-3, or AS-5) for 42 days and then biochemically modified,frozen, thawed, washed, and stored at 4 degrees C. in sodium chlorideand glucose solution for 24 hours,” Transfusion, 40: 1341-5 (2000))(hereby incorporated by reference in their entireties).

One approach that has proven successful in improving blood quality andextending its utility is through the depletion of oxygen and storageunder anaerobic conditions. Among the benefits of storing blood underoxygen depleted conditions are improved levels of ATP and 2,3-DPG, andreduced hemolysis. U.S. Pat. No. 5,624,794 to Bitensky et al., U.S. Pat.No. 6,162,396 to Bitensky et al., and U.S. Pat. No. 5,476,764 toBitensky (hereby incorporated by reference in their entireties) aredirected to the storage of red blood cells under oxygen-depletedconditions. U.S. Pat. No. 5,789,151 to Bitensky et al. is directed toblood storage additive solutions (hereby incorporated by reference inits entirety). U.S. Pat. No. 6,162,396 to Bitensky et al. (the '396patent) (hereby incorporated by reference in its entirety) disclosesanaerobic storage bags for blood storage that comprise an oxygenimpermeable outer layer, a red blood cell (RBCs) compatible inner layerthat is permeable to oxygen, and having an oxygen scrubber placedbetween the inner and outer layers.

Storing blood under oxygen depleted conditions can also result inreduced microparticle levels, reductions in the loss of deformability,reduced lipid and protein oxidation and higher post transfusion survivalwhen compared to blood stored under conventional conditions. See Yoshidaet al., “The effects of additive solution pH and metabolic rejuvenationon anaerobic storage of red cells,” Transfusion 48:2096-2105 (2008) andYoshida, T., et al. “Reduction of microparticle generation duringanaerobic storage of red blood cells. Transfusion”, 52, 83A (2012)(hereby incorporated by reference in their entireties). Anaerobicallystored RBCs further provide higher 24-hour in vivo recovery afterautologous transfusion, higher 2,3-DPG and ATP levels, lower hemolysis,and beneficial remodeling of metabolic pathway. See Reisz et al.“Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenaseregulate metabolic reprogramming of stored red blood cells,” Blood,128:e32-42 (2016); and Yoshida et al., “Extended storage of red bloodcells under anaerobic conditions,” Vox sanguinis 92:22-31 (2007) (herebyincorporated by reference in their entireties).

In the present disclosure, we demonstrate that oxygen reduced (OR) oroxygen and carbon dioxide reduced (OCR) blood from rats providesimproved ATP and 2,3-DPG during storage compared to conventionallystored blood, as has been previously demonstrated using human blood.Thus, OR or OCR rat RBCs are expected to have similar reductions inmicroparticles, improved deformability, reduced lipid and proteinoxidation and higher post transfusion survival.

Here we demonstrate for the first time that OR and OCR blood in ratsprovides for surprising improvements in clinical outcomes whentransfused to treat hemorrhagic trauma. Using a rat hemorrhagic shockresuscitation model, we show that OR or OCR RBCs provide for reducedorgan damage relative to conventionally stored blood. In addition, OR orOCR RBCs provide for reversal of the shock state using smaller pRBCvolumes. Finally, OR or OCR RBCs, when transfused to treat hemorrhagicshock more rapidly stabilized hemodynamics compared to conventionallystored pRBC of same storage duration.

OR and OCR RBCs provide for improved methods for treatment of traumaresulting in exsanguination to reduce mortality and morbidity overconventionally stored blood. OR and OCR RBCs provide for reduced organfailure, including reductions in levels of markers of lung and liverdamage. OR and OCR RBCs further provide reductions in the amounts ofblood necessary to restore and stabilize hemodynamic function. Thus, ORand OCR RBCs can provide for reducing the volume of RBCs required fortransfusion therapy when treating hemorrhagic trauma. The improvedquality of OR and OCR, in addition to the previously demonstratedimprovements to the ability of stored RBCs to deliver oxygen, alsoprovides for unexpected reductions in organ damage, morbidity, andmortality associated with trauma.

SUMMARY OF THE INVENTION

The present disclosure provides for, and includes, a method for treatinglow mean arterial pressure in a subject in need thereof comprisingproviding stored oxygen reduced blood having an oxygen saturation of 20%or less prior to and during storage, wherein the mean arterial pressurein the subject in need thereof is increased after providing the oxygenreduced blood to the subject in need thereof, and wherein the low meanarterial pressure is due to hemorrhagic trauma.

The present disclosure provides for, and includes, a method for reducingthe amount of blood needed for transfusion in a trauma patient in needthereof comprising providing oxygen reduced blood having an oxygensaturation of 20% or less prior to and during storage.

The present disclosure provides for, and includes, a method for reducinghemorrhagic shock in a trauma patient in need thereof comprisingproviding oxygen reduced blood having an oxygen saturation of 20% orless prior to and during storage, wherein the trauma patient comprises alactate level of between 0.5 and 2.5 millimole per liter (mmol/L) priorto the providing, and wherein the hemorrhagic shock is reversed.

The present disclosure provides for, and includes, a method of reducinga liver injury in a trauma patient in need of transfusion therapycomprising providing oxygen reduced blood having an oxygen saturation of20% or less prior to and during storage.

The present disclosure provides for, and includes, a method of reducingkidney failure in a hemorrhagic trauma patient in need of transfusiontherapy comprising providing oxygen reduced blood having an oxygensaturation of 20% or less prior to and during storage.

The present disclosure provides for, and includes, a method of reducinglung injury in a hemorrhagic trauma patient in need of transfusiontherapy comprising providing oxygen reduced blood having an oxygensaturation of 20% or less prior to and during storage.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is provided with reference to the accompanyingdrawings, wherein:

FIG. 1 is a graph presenting the results of an exemplary embodimentaccording to the present disclosure, comparing ATP levels inconventionally stored RBCs (unprocessed; control), sham control (SC),oxygen reduced RBCs (N₂; OR), and oxygen and carbon dioxide reduced RBCs(CO₂; OCR).

FIG. 2 is a graph presenting the results of an exemplary embodimentaccording to the present disclosure, comparing 2,3-DPG levels ofconventionally stored RBCs (unprocessed; control), sham control (SC),oxygen reduced RBCs (N₂; OR), and oxygen and carbon dioxide reduced RBCs(CO₂; OCR).

FIG. 3 is a graph presenting the results of an exemplary embodimentaccording to the present disclosure, presenting a comparison of thepercent recovery of control, sham, OR-RBCs, and OCR-RBCs transfused intoan animal.

FIGS. 4A and 4B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, presenting a comparisonof the percent hematocrit in animals resuscitated with control, OR-RBCs,and OCR-RBCs stored for 1 week (FIG. 4A) or 3 weeks (FIG. 4B). BL(baseline) identifies animals not under shock conditions. Shockidentifies animals under hemorrhagic shock. Early R identifies aresuscitation period of 60 mins. Late R identifies a resuscitationperiod of 60 mins.

FIGS. 5A and 5B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the mean arterial pressure (MAP) in animals resuscitated withcontrol, OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 5A) or 3 weeks(FIG. 5B). BL (baseline) identifies animals not under shock conditions.Shock identifies animals under hemorrhagic shock. Early R identifies aresuscitation period of 10 mins. Late R identifies a resuscitationperiod of 60 mins.

FIGS. 6A and 6B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the percent blood volume provided to animals during resuscitationafter 10, 20, 30, 45, and 60 mins. Control, OR-RBCs, and OCR-RBCs storedfor 1 week (FIG. 6A) or 3 weeks (FIG. 6B) are compared.

FIGS. 7A and 7B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of lactate in animals resuscitated with control, OR-RBCs,and OCR-RBCs stored for 1 week (FIG. 7A) or 3 weeks (FIG. 7B). BL(baseline) identifies animals not under shock conditions. Shockidentifies animals under hemorrhagic shock. Early R identifies aresuscitation period of 10 mins. Late R identifies a resuscitationperiod of 60 mins.

FIGS. 8A and 8B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of glucose in animals resuscitated with control, OR-RBCs,and OCR-RBCs stored for 1 week (FIG. 8A) or 3 weeks (FIG. 8B). BL(baseline) identifies animals not under shock conditions. Shockidentifies animals under hemorrhagic shock. Early R identifies aresuscitation period of 10 mins. Late R identifies a resuscitationperiod of 60 mins.

FIGS. 9A and 9B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of AST in animals resuscitated with control, OR-RBCs, andOCR-RBCs stored for 1 week (FIG. 9A) or 3 weeks (FIG. 9B).

FIGS. 10A and 10B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of ALT in animals resuscitated with control, OR-RBCs, andOCR-RBCs stored for 1 week (FIG. 10A) or 3 weeks (FIG. 10B).

FIGS. 11A and 11B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of serum creatinine in animals resuscitated with control,OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 11A) or 3 weeks (FIG.11B).

FIGS. 12A and 12B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of blood urea nitrogen (BUN) in animals resuscitated withcontrol, OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 12A) or 3 weeks(FIG. 12B).

FIGS. 13A and 13B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of CXCL1 in the liver of animals resuscitated withcontrol, OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 13A) or 3 weeks(FIG. 13B).

FIGS. 14A and 14B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of CXCL1 in the spleen of animals resuscitated withcontrol, OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 14A) or 3 weeks(FIG. 14B).

FIGS. 15A and 15B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of CXCL1 in the lungs of animals resuscitated withcontrol, OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 15A) or 3 weeks(FIG. 15B).

FIGS. 16A and 16B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of urinary neutrophil gelatinase-associated lipocalin(u-NGAL) in animals resuscitated with control, OR-RBCs, and OCR-RBCsstored for 1 week (FIG. 16A) or 3 weeks (FIG. 16B).

FIGS. 17A and 17B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the percentage of CD45+neutrophils in animals resuscitated withcontrol, OR-RBCs, and OCR-RBCs stored for 1 week (FIG. 17A) or 3 weeks(FIG. 17B).

FIGS. 18A and 18B are graphs presenting the results of an exemplaryembodiment according to the present disclosure, providing a comparisonof the amount of IL-6 in animals resuscitated with control, OR-RBCs, andOCR-RBCs stored for 1 week (FIG. 18A) or 3 weeks (FIG. 18B).

The examples set out herein illustrate(s) several embodiment(s) of thepresent disclosure but should not be construed as limiting the scope ofthe present disclosure in any manner.

DETAILED DESCRIPTION

Methods of the present disclosure provide for, and include, providing ahemorrhagic trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. Methodsalso provide for providing a hemorrhagic trauma patient with oxygenreduced stored blood that has an oxygen saturation of between 15 and 20%prior to and during storage. Methods also provide for providing ahemorrhagic trauma patient with oxygen reduced stored blood that has anoxygen saturation of between 10 and 15% prior to and during storage.Methods also provide for providing a hemorrhagic trauma patient withoxygen reduced stored blood that has an oxygen saturation of between 5and 10% prior to and during storage. Methods also provide for providinga hemorrhagic trauma patient with oxygen reduced stored blood that hasan oxygen saturation of between 3 and 5% prior to and during storage.

Methods also provide for providing oxygen reduced stored blood that hasan oxygen saturation of 20% or less prior to and during storage fortransfusion to a person having hemorrhagic shock. Methods also providefor providing oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage for transfusion to a personhaving hemorrhagic trauma. Also included are methods comprisingtransfusing oxygen reduced stored blood that has an oxygen saturation of20% or less prior to and during storage to a patient having an increasedrisk of trauma due to surgery. Methods providing oxygen reduced storedblood having an initial oxygen saturation of 20% or less includeproviding oxygen reduced stored blood having an initial oxygensaturation of 10% or less. Methods of providing oxygen reduced storedblood having an initial oxygen saturation of 20% or less further includeproviding oxygen reduced stored blood having an initial oxygensaturation of 5% or less. Methods of providing oxygen reduced storedblood having an initial oxygen saturation of 20% or less further includeproviding oxygen reduced stored blood having an initial oxygensaturation of 3% or less.

Methods of the present disclosure provide for, and include, providingoxygen reduced stored blood for the treatment of trauma having an oxygensaturation of 20% or less prior to and during storage for a storageperiod of at least one week, at least two weeks, at least 3 weeks, atleast 4 weeks, at least 5 weeks, or at least 6 weeks. Methods alsoprovide for providing oxygen reduced stored blood for the treatment oftrauma having an oxygen saturation of 15% or less after a storage periodof at least one week, at least two weeks, at least 3 weeks, at least 4weeks, at least 5 weeks, or at least 6 weeks. Methods also provide forproviding oxygen reduced stored blood for the treatment of trauma havingan oxygen saturation of 10% or less after a storage period of at leastone week, at least two weeks, at least 3 weeks, at least 4 weeks, atleast 5 weeks, or at least 6 weeks. Methods further provide forproviding oxygen reduced stored blood for the treatment of trauma havingan oxygen saturation of 5% or less after a storage period of at leastone week, at least two weeks, at least 3 weeks, at least 4 weeks, atleast 5 weeks, or at least 6 weeks. Methods further provide forproviding oxygen reduced stored blood for the treatment of trauma havingan oxygen saturation of 3% or less after a storage period of at leastone week, at least two weeks, at least 3 weeks, at least 4 weeks, atleast 5 weeks, or at least 6 weeks. Methods also provide for providingoxygen reduced stored blood for the treatment of trauma having an oxygensaturation of between 3 and 5% after a storage period of at least oneweek, at least two weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, or at least 6 weeks. Methods also provide for providing oxygenreduced stored blood for the treatment of trauma having an oxygensaturation of between 5 and 10% after a storage period of at least oneweek, at least two weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, or at least 6 weeks. Methods also provide for providing oxygenreduced stored blood for the treatment of trauma having an oxygensaturation of between 10 and 15% after a storage period of at least oneweek, at least two weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, or at least 6 weeks. Methods also provide for providing oxygenreduced stored blood for the treatment of trauma having an oxygensaturation of between 15 and 20% after a storage period of at least oneweek, at least two weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, or at least 6 weeks.

Methods of the present disclosure provide for, and include, providing atrauma patient with oxygen reduced stored blood that has an oxygensaturation of 20% or less prior to and during storage. In an aspect, atrauma patient suffers from a head trauma, a penetrating wound, bluntforce trauma, injury due to a fall, or injury due to a car accident. Inanother aspect, a trauma patient is a hemorrhagic trauma patient. In yetanother aspect, a trauma patient is hemorrhagic due to surgery, apenetrating wound, blunt force trauma, an injury due to a fall, or aninjury due to a car accident.

In an aspect of the present disclosure, a trauma patient or hemorrhagictrauma patient is a subject in need of OR and OCR stored blood. Inaspects of the present disclosure, a trauma patient is a hemorrhagictrauma patient in need of one or more units of blood as transfusiontherapy. In aspects of the present disclosure, a trauma patient is ahemorrhagic trauma patient in need of two or more units of blood astransfusion therapy. In aspects of the present disclosure, a traumapatient is a hemorrhagic trauma patient in need of three or more unitsof blood as transfusion therapy.

In an aspect of the present disclosure, a trauma patient is a patient inhemorrhagic shock. In an aspect, a trauma patient is in hemorrhagicshock due to a head trauma, a penetrating wound, blunt force trauma,injury from a fall, or injury from a car accident. In aspects of thepresent disclosure, a hemorrhagic trauma patient is a patient with aclass I hemorrhage. In another aspect, a hemorrhagic trauma patient is apatient with a class II hemorrhage. In another aspect, a hemorrhagictrauma patient is a patient with a class III hemorrhage. In anotheraspect, a hemorrhagic trauma patient is a patient with a class IVhemorrhage. In an aspect of the present disclosure, a hemorrhagic traumapatient loses up to 15% of blood volume. In another aspect, ahemorrhagic trauma patient loses between 15 and 30% of blood volume. Inanother aspect, a hemorrhagic trauma patient loses between 30 and 40% ofblood volume. In a further, a hemorrhagic trauma patient loses greaterthan 40% of blood volume.

The present disclosure provides for, and includes, a patient in need oftransfusion therapy with OR or OCR RBCs exhibits one or more signsselected from the group consisting of decreased mean arterial pressure,a decreased hematocrit, increased lactate, increased glucose, increasedaspartate aminotransferase (AST), increased alanine aminotransferase(ALT), increased urine neutrophil gelatinase-associated lipocalin(u-NGAL), increased serum creatinine, and increased blood urea nitrogen.In an aspect of the present disclosure, a patient in need of transfusiontherapy with OR or OCR RBCs is a hemorrhagic trauma patient having adecreased mean arterial pressure. The present disclosure provides for,and includes, a patient in need of transfusion therapy with OR or OCRRBCs exhibits increased aspartate aminotransferase (AST) and increasedalanine aminotransferase (ALT). The present disclosure provides for, andincludes, a patient in need of transfusion therapy with OR or OCR RBCsexhibits decreased mean arterial pressure and increased lactate. Thepresent disclosure provides for, and includes, a patient in need oftransfusion therapy with OR or OCR RBCs exhibits increased aspartateaminotransferase (AST), increased alanine aminotransferase (ALT), andincreased blood urea nitrogen. The present disclosure provides for, andincludes, a patient in need of transfusion therapy with OR or OCR RBCsexhibits increased aspartate aminotransferase (AST), increased alanineaminotransferase (ALT), increased serum creatinine, and increased bloodurea nitrogen. The present disclosure provides for, and includes, apatient in need of transfusion therapy with OR or OCR RBCs exhibitsincreased lactate and increased glucose. The present disclosure providesfor, and includes, a patient in need of transfusion therapy with OR orOCR RBCs exhibiting increased urine neutrophil gelatinase-associatedlipocalin (u-NGAL), increased serum creatinine, and increased blood ureanitrogen.

In another aspect, a patient in need of transfusion therapy with OR orOCR RBCs is a hemorrhagic trauma patient having a decreased hematocrit.In another aspect, a patient in need of transfusion therapy with OR orOCR RBCs is a hemorrhagic trauma patient having increased lactate. Inyet another aspect, a patient in need of transfusion therapy with OR orOCR RBCs is a hemorrhagic trauma patient having increased glucose. In afurther aspect, a hemorrhagic trauma patient having increased inaspartate aminotransferase (AST). In another aspect, a patient in needof transfusion therapy with OR or OCR RBCs is a hemorrhagic traumapatient having increased alanine aminotransferase (ALT). In anotheraspect, a patient in need of transfusion therapy with OR or OCR RBCs isa hemorrhagic trauma patient having increased urine neutrophilgelatinase-associated lipocalin (u-NGAL). In another aspect, a patientin need of transfusion therapy with OR or OCR RBCs is a hemorrhagictrauma patient having increased serum creatinine. In another aspect, apatient in need of transfusion therapy with OR or OCR RBCs is ahemorrhagic trauma patient having increased blood urea nitrogen.

In an aspect of the present disclosure, the OR and OCR stored blood foruse in transfusion therapy of a trauma patient in need thereof has aninitial oxygen saturation of 20% or less. In another aspect, OR and OCRstored blood has an initial oxygen saturation of 10% or less. In anotheraspect, OR and OCR stored blood has an initial oxygen saturation of 5%or less. In another aspect, OR and OCR stored blood has an initialoxygen saturation of 3% or less.

In an aspect of the present disclosure, the OCR stored blood for use intransfusion therapy of a trauma patient in need thereof has an initialpCO₂ (at 37° C.) of between 10 and 40 mmHg. In another aspect, OCRstored blood has an initial pCO₂ of between 10 and 30 mmHg. In anotheraspect, OCR stored blood has an initial pCO₂ of between 10 and 20 mmHg.In another aspect, OCR stored blood has an initial pCO₂ of between 10and 15 mmHg. In yet another aspect, OCR stored blood has an initial pCO₂of less than 10 mmHg.

In an aspect of the present disclosure, OR and OCR stored blood for usein transfusion therapy of a trauma patient in need thereof has aninitial oxygen saturation of 20% or less and is stored for less than 2days. In an aspect, OR and OCR stored blood has an initial oxygensaturation of 20% or less is stored for less than 7 days. In anotheraspect, OR and OCR stored blood has an initial oxygen saturation of 20%or less is stored for less than 14 days. In another aspect, oxygenreduced stored blood has an initial oxygen saturation of 20% or less isstored for less than 21 days. In another aspect, oxygen reduced storedblood for use in transfusion therapy of a trauma patient in need thereofhas an initial oxygen saturation of 20% or less is stored for less than28 days. In another aspect, oxygen reduced stored blood has an initialoxygen saturation of 20% or less is stored for less than 35 days. Inanother aspect, oxygen reduced stored blood has an initial oxygensaturation of 20% or less is stored for less than 42 days. In anotheraspect, oxygen reduced stored blood has an initial oxygen saturation of20% or less is stored for less than 45 days. In an aspect of the presentdisclosure, OR and OCR stored blood has an oxygen saturation of 20% orless during storage.

Suitable blood for use methods according to the present disclosure foruse in transfusion therapy of a trauma patient in need thereof compriseoxygen reduced stored blood having an anticoagulant. In an aspect of thepresent disclosure, oxygen reduced red blood cells is stored for up to 3weeks to produce oxygen reduced stored blood. In another aspect, oxygenreduced stored blood usually further comprise an additive solution.Suitable additive 5 solutions according to the present disclosureinclude AS-1, AS-3 (Nutricel®), AS-5, SAGM, PAGG-SM, PAGG-GM, MAP, AS-7,ESOL-5, EAS61, OFAS1, OFAS3, and combinations thereof. In an aspect, theadditive solution is added at the time of component separation. In anaspect, the additive solution is AS-1. In another aspect, the additivesolution is AS-3. In other aspects, the additive solution is SAGM.

Methods of the present disclosure provide for, and include, increasingthe mean arterial pressure (MAP) in a hemorrhagic trauma patient in needof transfusion therapy comprising providing a trauma patient with oxygenreduced stored blood that has an oxygen saturation of 20% or less priorto and during storage. In an aspect, the mean arterial pressure isincreased by between 20 and 60%. In another aspect, the mean arterialpressure is increased by between 30 and 60%. In another aspect, the meanarterial pressure of a trauma patient receiving transfusion therapy ofOR or OCR blood is increased by between 30 and 50%. In yet anotheraspect, the mean arterial pressure is increased by between 30 and 60%.In a further aspect, the mean arterial pressure of a trauma patientreceiving transfusion therapy of OR or OCR blood is increased by between30 and 40%. In an aspect, the mean arterial pressure is increased by atleast 10, 20, 30, 40, 50, 60, 70, 80, or 90% more than the mean arterialpressure of a patient transfused with conventionally stored blood.

In an aspect of the present disclosure, the mean arterial pressure isincreased by at least 1.5 fold. In another aspect, the mean arterialpressure of a trauma patient receiving transfusion therapy of OR or OCRblood is increased by at least 2 fold. In a further aspect, the meanarterial pressure is increased by between 1 and 2 fold. In an aspect ofthe present disclosure, the mean arterial pressure of a trauma patientreceiving transfusion therapy of OR or OCR blood is increased by atleast 10 mmHg, at least 20 mmHg, at least 30 mmHg, at least 40 mmHg, atleast 50 mmHg, or at least 60 mmHg. In another aspect, the mean arterialpressure of a trauma patient receiving transfusion therapy of OR or OCRblood is increased by between 20 and 50 mmHg. In a further aspect, themean arterial pressure is increased by between 30 and 50 mmHg.

Methods of the present disclosure provide for, and include, increasingthe mean arterial pressure in a trauma patient in need of transfusiontherapy to between 70 and 110 mmHg comprising providing a trauma patientwith oxygen reduced stored blood that has an oxygen saturation of 20% orless prior to, and during storage. In another aspect, the mean arterialpressure of a trauma patient receiving transfusion therapy of OR or OCRblood is increased to at least 70 mmHg. In another aspect, the meanarterial pressure of a trauma patient receiving transfusion therapy ofOR or OCR blood is increased to at least 80 mmHg.

In yet another aspect, the mean arterial pressure is increased to atleast 90 mmHg. In a further aspect, the mean arterial pressure isincreased to at least 100 mmHg. In an aspect of the present disclosure,the mean arterial pressure in a subject in need thereof remains between70 and 110 mmHg for at least 1 hour after transfusion. In anotheraspect, the mean arterial pressure remains between 70 and 110 mmHg forat least 2 hours after transfusion. In yet another aspect, the meanarterial pressure remains between 70 and 105 mmHg for at least 3 hoursafter transfusion. In another aspect, the mean arterial pressure remainsbetween 70 and 110 mmHg for at least 4 hours after transfusion. Inanother aspect, the mean arterial pressure remains between 70 and 110mmHg for at least 5 hours after transfusion.

Methods of the present disclosure provide for, and include, increasingthe mean arterial pressure in a trauma patient in need of transfusiontherapy at a rate faster than the mean arterial pressure of a patienttransfused with conventionally stored blood comprising providing atrauma patient with oxygen reduced stored blood that has an oxygensaturation of 20% or less prior to and during storage. In an aspect, themean arterial pressure of a patient transfused with OR or OCR blood isrestored to within normal physiologic parameters in half the time whencompared to conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing theamount of stored blood needed for transfusion in a hemorrhagic traumapatient in need of transfusion therapy comprising providing a traumapatient with oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage. In an aspect, the amount ofOR stored blood needed for transfusion is between 10 and 90% less thanthe amount of conventionally stored blood needed. In another aspect, theamount of OR stored blood needed for transfusion is between 10 and 30%less than the amount of conventionally stored blood needed. In anotheraspect, the amount of OR stored blood needed for transfusion is between20 and 50% less than the amount of conventionally stored blood needed.In another aspect, the amount of OR stored blood needed for transfusionis between 20 and 80% less than the amount of conventionally storedblood needed. In another aspect, the amount of OR stored blood neededfor transfusion is between 30 and 80% less than the amount ofconventionally stored blood needed. In yet another aspect, the amount ofOR stored blood needed for transfusion is between 40 and 85% less thanthe amount of conventionally stored blood needed. In a further aspect,the amount of OR stored blood needed for transfusion is between 50 and90% less than the amount of conventionally stored blood needed.

Methods of the present disclosure provide for, and include, reducing theamount of stored blood needed for transfusion in a hemorrhagic traumapatient in need of transfusion therapy by at least 10% less comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. In anaspect, the amount of OR stored blood needed for transfusion is at least20% less than the amount of conventionally stored blood needed. Inanother aspect, the amount of OR stored blood needed for transfusion isat least 30% less than the amount of conventionally stored blood needed.In another aspect, the amount of OR stored blood needed for transfusionis at least 40% less than the amount of conventionally stored bloodneeded. In another aspect, the amount of OR stored blood needed fortransfusion is at least 50% less than the amount of conventionallystored blood needed. In yet another aspect, the amount of OR storedblood needed for transfusion is at least 60% less than the amount ofconventionally stored blood needed. In another aspect, the amount of ORstored blood needed for transfusion is at least 70% less than the amountof conventionally stored blood needed. In a further aspect, the amountof OR stored blood needed for transfusion is between about 10 and 20%,about 20 and 30%, about 30 and 40%, about 40 and 50%, about 50 and 60%,about 60 and 70%, about 70 and 80%, about 80 and 90%, or about 90 and95% less than the amount of conventionally stored blood needed. Inanother aspect, the amount of OR stored blood needed for transfusion ina trauma patient in need of transfusion therapy is between 10 and 20%,20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70 and 80%,80 and 90%, or 90 and 95% less than the amount of conventionally storedblood needed.

Lactate clearance is a biomarker for resuscitation from hemorrhagicshock. See Hashmi et al., “Predictors of mortality in geriatric traumapatients: a systematic review and meta-analysis,” The journal of traumaand acute care surgery, 76:894-901 (2014); Regnier et al., “Prognosticsignificance of blood lactate and lactate clearance in trauma patients,”Anesthesiology, 117:1276-88 (2012); and Zhang et al., “Lactate clearanceis a useful biomarker for the prediction of all-cause mortality incritically ill patients: a systematic review and meta-analysis,”Critical care medicine, 42:2118-25 (2014) (“Zhang 2014”) (herebyincorporated by reference in their entireties). The clinical value oflactate clearance is useful in predicting the outcome of patients withseptic shock and critically ill patients without evident circulatoryshock. Elevated lactate is an indicator of adverse clinical outcome, andits rapid clearance is universally associated with improved outcome inheterogeneous ICU or ED patient population. See Zhang 2014. Decreasedlactate levels in 5 animals resuscitated with OR-RBCs compared toconventionally RBCs support the notion that resuscitation with OR RBCscan significantly improve patients' clinical outcome. See FIGS. 7A and7B.

Methods of the present disclosure provide for, and include, reducing thelactate level in a trauma patient in need of transfusion therapycomprising providing a trauma patient with oxygen reduced (OR) storedblood that has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the lactate level is reduced by between 10 and90%. In an aspect, transfusion with OR stored blood reduces the lactatelevel in a trauma patient in need of transfusion therapy by between 10and 50%. In another aspect, transfusion with OR stored blood reduces thelactate level in a trauma patient in need of transfusion therapy bybetween 20 and 40%. In another aspect, transfusion with OR stored bloodreduces the lactate level in a trauma patient in need of transfusiontherapy by between 50 and 90%. In yet another aspect, transfusion withOR stored blood reduces the lactate level in a trauma patient in need oftransfusion therapy by between 60 and 90%. In another aspect,transfusion with OR stored blood reduces the lactate level in a traumapatient in need of transfusion therapy by between 10 and 20%, 20 and30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70 and 80%, or 80and 90%. In another aspect, transfusion with OR stored blood reduces thelactate level in a trauma patient in need of transfusion therapy by atleast 10%. In another aspect, transfusion with OR stored blood reducesthe lactate level in a trauma patient in need of transfusion therapy byat least 20%. In a further aspect, transfusion with OR stored bloodreduces the lactate level in a trauma patient in need of transfusiontherapy by at least 30, at least 40, at least 50, at least 60, at least70, at least 80, or at least 90%.

Methods of the present disclosure provide for, and include, reducingelevated lactate levels in a trauma patient in need of transfusiontherapy to between about 0.5 and about 2.5 mmol/L comprising providing atrauma patient with oxygen reduced stored blood that has an oxygensaturation of 20% or less prior to and during storage. In an aspect, thelactate level in a trauma patient in need of transfusion therapy isreduced to between about 0.9 and about 2 mmol/L. In an aspect, thelactate level in a trauma patient in need of transfusion therapy isreduced to between about 0.9 and about 1.7 mmol/L. In another aspect,the lactate level in a patient in need of transfusion therapy is reducedto between about 1.4 and about 2.4 mmol/L. In another aspect, thelactate level in a trauma patient in need of transfusion therapy isreduced to between about 1.7 and about 2.5 mmol/L. In yet anotheraspect, the lactate level in a trauma patient in need of transfusiontherapy is reduced to less than about 2.5 mmol/L. In a further aspect,the lactate level in a trauma patient in need of transfusion therapy isreduced to less than about 2.0 mmol/L. In another aspect, the lactatelevel in a trauma patient in need of transfusion therapy is reduced toless than about 1.5 mmol/L. In another aspect, the lactate level in atrauma patient in need of transfusion therapy is reduced to less thanabout 1.0 mmol/L. In yet another aspect, the lactate level in a traumapatient in need of transfusion therapy is reduced to between about 0.5and about 1.0 mmol/L.

Methods of the present disclosure provide for, and include, reducingelevated lactate levels in a trauma patient in need of transfusiontherapy to between 0.5 and 2.5 mmol/L comprising providing a traumapatient with oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage. In an aspect, the lactatelevel in a patient in need of transfusion therapy is reduced to between0.9 and 2 mmol/L. In an aspect, the lactate level in a trauma patient inneed of transfusion therapy is reduced to between 0.9 and 1.7 mmol/L. Inanother aspect, the lactate level in a trauma patient in need oftransfusion therapy is reduced to between 1.4 and 2.4 mmol/L. In anotheraspect, the lactate level in a trauma patient in need of transfusiontherapy is reduced to between 1.7 and 2.5 mmol/L. In another aspect, thelactate level in a trauma patient in need of transfusion therapy isreduced to between 0.5 and 1 mmol/L.

Methods of the present disclosure provide for, and include, reducingelevated lactate levels in a hemorrhagic trauma patient in need oftransfusion therapy to less than 4 mmol/L comprising providing a traumapatient with oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage. In an aspect, the lactatelevel in a trauma patient in need of transfusion therapy is reduced toless than 3 mmo1/L. In yet another aspect, the lactate level in a traumapatient in need of transfusion therapy is reduced to less than 2.5mmol/L. In another aspect, the lactate level in a patient is reduced toless than 2.3 mmol/L. In another aspect, the lactate level in a traumapatient in need of transfusion therapy is reduced to less than 2 mmol/L.In another aspect, the lactate level in a trauma patient in need oftransfusion therapy is reduced to less than 2 mmol/L. In another aspect,the lactate level in a trauma patient in need of transfusion therapy isreduced to less than 1.5 mmol/L. In another aspect, the lactate level ina trauma patient in need of transfusion therapy is reduced to less than1 mmol/L.

Blood glucose level is also known to be a predictor for outcome inseveral disease patterns and particularly in trauma patients. Traumapatients are more prone to poor outcome due to hyperglycemia than othercritically ill patients. See Kreutziger et al., “Admission blood glucosepredicted hemorrhagic shock in multiple trauma patients,” Injury,46:15-20 (2015) (hereby incorporated by reference in its entirety).Studies evaluating the relationship of early hyperglycemia and traumapatients examined early hyperglycemia at three possible cutoffs: glucose≥110 mg/dL, glucose≥150 mg/dL, and glucose≥200 mg/dL. See Laird et al.,“Relationship of early hyperglycemia to mortality in trauma patients,” JTrauma, 56:1058-62 (2004) (hereby incorporated by reference in itsentirety). A glucose level; ≥200 mg/dL, is associated with significantlyhigher infection and mortality rates in trauma patients independent ofinjury characteristics. This was not true at the cutoffs of ≥110 mg/dLor ≥150 mg/dL. Decreased glucose levels in animals resuscitated with OR-and OCR-RBCs compared to conventionally RBCs support the notion thatresuscitation with OR-RBCs can significantly improve patients' clinicaloutcome. See FIGS. 8A and 8B.

Methods of the present disclosure provide for, and include, reducingglucose in a trauma patient in need of transfusion therapy comprisingproviding a trauma patient with oxygen reduced (OR) stored blood thathas an oxygen saturation of 20% or less prior to and during storage. Inan aspect, glucose is reduced by between 10 and 90% as compared totransfusion of blood stored under conventional conditions. In an aspect,transfusion with OR stored blood reduces glucose by between 10 and 50%as compared to transfusion of blood stored under conventionalconditions. In another aspect, transfusion with OR stored blood reducesglucose by between 20 and 40% as compared to transfusion of blood storedunder conventional conditions. In another aspect, transfusion with ORstored blood reduces glucose by between 50 and 90% as compared totransfusion of blood stored under conventional conditions. In yetanother aspect, transfusion with OR stored blood reduces glucose bybetween 60 and 90%. In another aspect, transfusion with OR stored bloodreduces glucose by between 10 and 20%, 20 and 30%, 30 and 40%, 40 and50%, 50 and 60%, 60 and 70%, 70 and 80%, or 80 and 90% as compared totransfusion of blood stored under conventional conditions. In anotheraspect, transfusion with OR stored blood reduces glucose by at least 10%as compared to transfusion of blood stored under conventionalconditions. In another aspect, transfusion with OR stored blood reducesglucose by at least 20%. In a further aspect, transfusion with OR storedblood reduces glucose by at least 30, at least 40, at least 50, at least60, at least 70, at least 80, or at least 90%.

Methods of the present disclosure provide for, and include, reducingglucose levels in a trauma patient in need of transfusion therapy tobetween about 70 and about 120 mg/dL comprising providing a traumapatient with oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage. In an aspect, glucose in apatient after transfusion therapy with OR or OCR blood is between about70 and about 110 mg/dL. In another aspect, glucose in a patient aftertransfusion therapy with OR or OCR blood is between about 70 and about100 mg/dL. In another aspect, glucose in a trauma patient aftertransfusion therapy with OR or OCR blood is between about 90 and about120 mg/dL. In another aspect, glucose in a trauma patient aftertransfusion therapy with OR or OCR blood is between about 90 and about100 mg/dL.

Methods of the present disclosure provide for, and include, reducingglucose levels in a trauma patient in need of transfusion therapy tobetween 70 and 120 mg/dL comprising providing a trauma patient withoxygen reduced stored blood that has an oxygen saturation of 20% or lessprior to and during storage. In an aspect, glucose in a patient aftertransfusion therapy with OR or OCR blood is between 70 and 110 mg/dL. Inanother aspect, glucose in a patient is between 70 and 100 mg/dL. Inanother aspect, glucose in a patient is between 90 and 120 mg/dL. Inanother aspect, glucose in a patient after transfusion therapy with ORor OCR blood is between 90 and 100 mg/dL.

Methods of the present disclosure provide for, and include, reducingglucose levels in a trauma patient in need of transfusion therapy toless than 120 mg/dL comprising providing a trauma patient with oxygenreduced stored blood that has an oxygen saturation of 20% or less priorto and during storage. In a further aspect, glucose in a patient aftertransfusion therapy with OR or OCR blood is less than 110 mmol/L. In yetanother aspect, glucose in a patient after transfusion therapy with ORor OCR blood is less than 100 mg/dL. In another aspect, glucose in apatient after transfusion therapy with OR or OCR blood is less than 200mg/dL. In another aspect, glucose in a patient after transfusion therapywith OR or OCR blood is less than 90 mg/dL. In another aspect, glucosein a patient after transfusion therapy with OR or OCR blood is less than80 mg/dL.

In an aspect of the present disclosure, a trauma patient is at increasedrisk of complications from transfusion therapies based on a pre-existingor underlying condition. In an aspect, a trauma patient has apre-existing or underlying condition selected from the group consistingof a diabetes, ischemic heart disease, systemic inflammatory syndromebrought on by trauma or infection, multiple organ failure brought on bytrauma or infection, smoke inhalation, chronic pulmonary obstructivedisease such as systemic inflammation due to infection, a coagulopathydisorder, and autoimmune diseases. In another aspect, a trauma patienthas one or more pre-existing or underlying conditions selected from thegroup consisting of a diabetes, ischemic heart disease, systemicinflammatory syndrome brought on by trauma or infection, multiple organfailure brought on by trauma or infection, smoke inhalation, and chronicpulmonary obstructive disease such as systemic inflammation due toinfection, a coagulopathy disorder, and autoimmune diseases. In anotheraspect, a trauma patient has two or more pre-existing or underlyingconditions selected from the group consisting of a diabetes, ischemicheart disease, systemic inflammatory syndrome brought on by trauma orinfection, multiple organ failure brought on by trauma or infection,smoke inhalation, chronic pulmonary obstructive disease such as systemicinflammation due to infection, a coagulopathy disorder, and autoimmunediseases. In another aspect, a trauma patient has three or morepre-existing or underlying conditions selected from the group consistingof a diabetes, ischemic heart disease, systemic inflammatory syndromebrought on by trauma or infection, multiple organ failure brought on bytrauma or infection, smoke inhalation, chronic pulmonary obstructivedisease such as systemic inflammation due to infection, a coagulopathydisorder, and autoimmune diseases.

During hemorrhagic shock, patients experience an adverse event includingliver damage or failure, kidney damage or failure, lung damage orfailure, or a combination thereof. The present disclosure provides for,and includes, a patient in need of transfusion therapy with OR or OCRRBCs exhibits one or more adverse event selected from the groupconsisting of liver damage or failure, kidney damage or failure, or lungdamage or failure. The present disclosure provides for, and includes, apatient in need of transfusion therapy with OR or OCR RBCs exhibits twoor more adverse event selected from the group consisting of liver damageor failure, kidney damage or failure, or lung damage or failure.

Methods of the present disclosure provide for, and include, reducing anadverse event in a trauma patient comprising providing a trauma patientin need of transfusion therapy with oxygen reduced stored blood that hasan oxygen saturation of 20% or less prior to and during storage. In anaspect, the adverse event after transfusion therapy with OR or OCR bloodis reduced by at least 5%. In another aspect, the adverse event aftertransfusion therapy with OR or OCR blood is reduced by at least 10%. Inanother aspect, the adverse event after transfusion therapy with OR orOCR blood is reduced by at least 20%. In another aspect, the adverseevent after transfusion therapy with OR or OCR blood is reduced by atleast 30%. In another aspect, the adverse event after transfusiontherapy with OR or OCR blood is reduced by at least 40%. In anotheraspect, the adverse event after transfusion therapy with OR or OCR bloodis reduced by at least 50%. In another aspect, the adverse event aftertransfusion therapy with OR or OCR blood is reduced by at least 60%. Inanother aspect, the adverse event is reduced by at least 70%. In anotheraspect, the adverse event after transfusion therapy with OR or OCR bloodis reduced by at least 80%. In another aspect, the adverse event isreduced by at least 90%. In a further aspect, the adverse event aftertransfusion therapy with OR or OCR blood is reduced by between 1 and10%, 10 and 20%, 20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and70%, 70 and 80%, 80 and 90%, or 90 and 95%. In an aspect, the adverseevent after transfusion therapy with OR or OCR blood is liver injury ordamage. In another aspect, the adverse event is lung injury or damage.In yet another aspect, the adverse event is kidney injury or damage. Ina further aspect, an adverse event is liver injury, lung injury, kidneyinjury, or a combination thereof

Elevated levels of liver enzymes, including but not limited to aspartateaminotransferase (AST) and alanine aminotransferase (ALT), signify someform of liver damage, shock, or injury. Methods of the presentdisclosure provide for, and include, reducing elevated levels of liverenzymes in a trauma patient comprising providing a trauma patient withoxygen reduced stored blood that has an oxygen saturation of 20% or lessprior to and during storage.

Methods of the present disclosure provide for, and include, reducing ASTlevels in a trauma patient in need of transfusion therapy comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. In anaspect, the AST level is reduced by at least 5% relative to the ASTlevel of a patient transfused with conventionally stored blood. Inanother aspect, the AST level is reduced by at least 10% relative to theAST level of a patient transfused with conventionally stored blood. Inanother aspect, the AST level is reduced by at least 20% relative to theAST level of a patient transfused with conventionally stored blood. Inanother aspect, the AST level is reduced by at least 30% relative to theAST level of a patient transfused with conventionally stored blood. Inanother aspect, the AST level is reduced by at least 40%. In anotheraspect, the AST level is reduced by at least 50% relative to the ASTlevel of a patient transfused with conventionally stored blood. Inanother aspect, the AST level is reduced by at least 60%. In anotheraspect, the AST level is reduced by at least 70% relative to the ASTlevel of a patient transfused with conventionally stored blood. In yetanother aspect, the AST level is reduced by at least 80%. In a furtheraspect, the AST level is reduced by at least 90% relative to the ASTlevel of a patient transfused with conventionally stored blood. In afurther aspect, the AST level is reduced by between 1 and 10%, 10 and20%, 20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70 and80%, 80 and 90%, or 90 and 95% relative to the AST level of a patienttransfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing ASTlevels in a trauma patient in need of transfusion therapy by between 1.5and 10 fold comprising providing a trauma patient with oxygen reducedstored blood that has an oxygen saturation of 20% or less prior to andduring storage. In an aspect, the AST level is reduced by between 2 and3 fold relative to the AST level of a patient transfused withconventionally stored blood. In another aspect, the AST level is reducedby between 3 and 4 fold. In another aspect, the AST level is reduced bybetween 4 and 10 fold. In another aspect, the AST level is reduced bybetween 6 and 9 fold relative to the AST level of a patient transfusedwith conventionally stored blood. In a further aspect, the AST level isreduced by between 2 and 5 fold. In another aspect, the AST level isreduced by between 10 and 50 fold relative to the AST level of a patienttransfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing ASTlevels in a trauma patient in need of transfusion therapy by at least1.5 fold comprising providing a trauma patient with oxygen reducedstored blood that has an oxygen saturation of 20% or less prior to andduring storage. In an aspect, the AST level is reduced by at least 2fold relative to the AST level of a patient transfused withconventionally stored blood. In another aspect, the AST level is reducedby at least 3 fold relative to the AST level of a patient transfusedwith conventionally stored blood. In another aspect, the AST level isreduced by at least 4 fold relative to the AST level of a patienttransfused with conventionally stored blood. In another aspect, the ASTlevel is reduced by at least 5 fold relative to the AST level of apatient transfused with conventionally stored blood. In a furtheraspect, the AST level is reduced by at least 6 fold. In another aspect,the AST level is reduced by at least 7 fold relative to the AST level ofa patient transfused with conventionally stored blood. In anotheraspect, the AST level is reduced by at least 8 fold. In another aspect,the AST level is reduced by at least 9 fold relative to the AST level ofa patient transfused with conventionally stored blood. In anotheraspect, the AST level is reduced by at least 10 fold relative to the ASTlevel of a patient transfused with conventionally stored blood. In afurther aspect, the AST level is reduced by at least 50 fold relative tothe AST level of a patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing ALTlevels in a trauma patient in need of transfusion therapy comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. In anaspect, the ALT level is reduced by at least 5% relative to the ALTlevel of a patient transfused with conventionally stored blood. Inanother aspect, the ALT level is reduced by at least 10% relative to theALT level of a patient transfused with conventionally stored blood. Inanother aspect, the ALT level is reduced by at least 20% relative to theALT level of a patient transfused with conventionally stored blood. Inanother aspect, the ALT level is reduced by at least 30% relative to theALT level of a patient transfused with conventionally stored blood. Inanother aspect, the ALT level is reduced by at least 40% relative to theAS ALT T level of a patient transfused with conventionally stored blood.In another aspect, the ALT level is reduced by at least 50% relative tothe ALT level of a patient transfused with conventionally stored blood.In another aspect, the ALT level is reduced by at least 60% relative tothe ALT level of a patient transfused with conventionally stored blood.In another aspect, the ALT level is reduced by at least 70% relative tothe ALT level of a patient transfused with conventionally stored blood.In yet another aspect, the ALT level is reduced by at least 80% relativeto the ALT level of a patient transfused with conventionally storedblood. In a further aspect, the ALT level is reduced by at least 90%. Ina further aspect, the ALT level is reduced by between 1 and 10%, 10 and20%, 20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70 and80%, 80 and 90%, or 90 and 95% relative to the ALT level of a patienttransfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing ALTlevels in a trauma patient in need of transfusion therapy by between 1.5and 10 fold comprising providing a trauma patient with oxygen reducedstored blood that has an oxygen saturation of 20% or less prior to andduring storage. In an aspect, the ALT level is reduced by between 2 and3 fold relative to the ALT level of a patient transfused withconventionally stored blood.

In another aspect, the ALT level is reduced by between 3 and 4 foldrelative to the ALT level of a patient transfused with conventionallystored blood. In another aspect, the ALT level is reduced by between 4and 10 fold relative to the ALT level of a patient transfused withconventionally stored blood. In another aspect, the ALT level is reducedby between 6 and 9 fold relative to the ALT level of a patienttransfused with conventionally stored blood. In a further aspect, theALT level is reduced by between 2 and 5 fold. In another aspect, the ALTlevel is reduced by between 10 and 50 fold relative to the ALT level ofa patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing ALTlevels in a trauma patient in need of transfusion therapy by at least1.5 fold comprising providing a trauma patient with oxygen reducedstored blood that has an oxygen saturation of 20% or less prior to andduring storage. In an aspect, the ALT level is reduced by at least 2fold relative to the ALT level of a patient transfused withconventionally stored blood. In another aspect, the ALT level is reducedby at least 3 fold relative to the ALT level of a patient transfusedwith conventionally stored blood. In another aspect, the ALT level isreduced by at least 4 fold relative to the ALT level of a patienttransfused with conventionally stored blood. In another aspect, the ALTlevel is reduced by at least 5 fold relative to the ALT level of apatient transfused with conventionally stored blood. In a furtheraspect, the ALT level is reduced by at least 6 fold relative to the ALTlevel of a patient transfused with conventionally stored blood. Inanother aspect, the ALT level is reduced by at least 7 fold relative tothe ALT level of a patient transfused with conventionally stored blood.In another aspect, the ALT level is reduced by at least 8 fold relativeto the ALT level of a patient transfused with conventionally storedblood. In another aspect, the ALT level is reduced by at least 9 foldrelative to the ALT level of a patient transfused with conventionallystored blood. In another aspect, the ALT level is reduced by at least 10fold relative to the ALT level of a patient transfused withconventionally stored blood. In a further aspect, the ALT level isreduced by at least 50 fold relative to the ALT level of a patienttransfused with conventionally stored blood.

Markers of kidney function during and after hemorrhagic trauma includeurine neutrophil gelatinase-associated lipocalin (u-NGAL), serumcreatinine, and blood urea nitrogen (BUN). See Treeprasertsuk et al.,“Urine neutrophil gelatinase-associated lipocalin: a diagnostic andprognostic marker for acute kidney injury (AKI) in hospitalizedcirrhotic patients with AKI-prone conditions,” BMC Gastroenterol 15:140(2015) (hereby incorporated by reference in its entirety). Geneexpression analyses reported in greater than 150 distinct studiesperformed in AKI models from several species ranging from rodents tohumans have consistently revealed the NGAL gene to be one of the mostdramatically upregulated genes in the kidney soon after an ischemic or anephrotoxic insult. See Ciccia et al., “Pediatric acute kidney injury:prevalence, impact and management challenges,” Int J Nephrol RenovascDis, 10:77-84 (2017) (hereby incorporated by reference in its entirety).Similarly serum creatinine levels can vary depending on age, race andbody size, however, rising creatinine levels are indicative of kidneydamage. Creatinine levels of greater than 1.2 for women and greater than1.4 for men may be an early sign of kidney damage. Increased blood ureanitrogen (BUN) is seen associated with kidney disease or failure, aswell as, congestive heart failure, shock and bleeding in the digestivetract. If the BUN level is higher than 100 mg/dL it points to severekidney damage. Decreased levels of BUN are also a concern and can pointto fluid excess, trauma, surgery, opioids, malnutrition, and anabolicsteroid use. See Pagana, “Mosby's Manual of Diagnostic and LaboratoryTests,” St. Louis Mosby, Inc., (1998); and Gowda, et al., “Markers ofrenal function tests,” N Am J Med Sci. 2(4): 170-173(2010) (herebyincorporated by reference in their entireties). Decreased u-NGAL (FIGS.16A and 16B) serum creatinine (FIGS. 11A and 11B), and BUN (FIGS. 12Aand 12B) levels in animals resuscitated with OR- and OCR-RBCs comparedto conventionally RBCs, as provided by the present disclosure, show thatresuscitation with OR-RBCs can significantly improve patients' clinicaloutcome.

Methods of the present disclosure provide for, and include, reducingurinary neutrophil gelatinase-associated lipocalin (u-NGAL) levels in atrauma patient in need of transfusion therapy comprising providing atrauma patient with oxygen reduced stored blood that has an oxygensaturation of 20% or less prior to and during storage. In an aspect, theu-NGAL level is reduced by at least 5% relative to the u-NGAL level of apatient transfused with conventionally stored blood. In another aspect,the u-NGAL level is reduced by at least 10% relative to the u-NGAL levelof a patient transfused with conventionally stored blood. In anotheraspect, the u-NGAL level is reduced by at least 20% relative to theu-NGAL level of a patient transfused with conventionally stored blood.In another aspect, the u-NGAL level is reduced by at least 30%. Inanother aspect, the u-NGAL level is reduced by at least 40% relative tothe u-NGAL level of a patient transfused with conventionally storedblood. In another aspect, the u-NGAL level is reduced by at least 50%relative to the u-NGAL level of a patient transfused with conventionallystored blood. In another aspect, the u-NGAL level is reduced by at least60% relative to the u-NGAL level of a patient transfused withconventionally stored blood. In another aspect, the u-NGAL level isreduced by at least 70% relative to the u-NGAL level of a patienttransfused with conventionally stored blood. In yet another aspect, theu-NGAL level is reduced by at least 80% relative to the u-NGAL level ofa patient transfused with conventionally stored blood. In a furtheraspect, the u-NGAL level is reduced by at least 90% relative to theu-NGAL level of a patient transfused with conventionally stored blood.In a further aspect, the u-NGAL level is reduced by between 1 and 10%,10 and 20%, 20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%,70 and 80%, 80 and 90%, or 90 and 95% relative to the u-NGAL level of apatient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducingurinary neutrophil gelatinase-associated lipocalin (u-NGAL) levels in atrauma patient in need of transfusion therapy comprising providing atrauma patient by between 1.5 and 10 fold with oxygen reduced storedblood that has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the u-NGAL level is reduced by between 2 and 3fold relative to the u-NGAL level of a patient transfused withconventionally stored blood. In another aspect, the u-NGAL level isreduced by between 3 and 4 fold relative to the u-NGAL level of apatient transfused with conventionally stored blood. In another aspect,the u-NGAL level is reduced by between 4 and 10 folds. In anotheraspect, the u-NGAL level is reduced by between 6 and 9 fold relative tothe u-NGAL level of a patient transfused with conventionally storedblood. In a further aspect, the u-NGAL level is reduced by between 2 and5 fold relative to the u-NGAL level of a patient transfused withconventionally stored blood. In another aspect, the u-NGAL level isreduced by between 10 and 50 fold relative to the u-NGAL level of apatient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducingurinary neutrophil gelatinase-associated lipocalin (u-NGAL) levels in atrauma patient in need of transfusion therapy comprising providing atrauma patient by at least 1.5 fold with oxygen reduced stored bloodthat has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the u-NGAL level is reduced by at least 2 foldrelative to the u-NGAL level of a patient transfused with conventionallystored blood. In another aspect, the u-NGAL level is reduced by at least3 fold relative to the u-NGAL level of a patient transfused withconventionally stored blood. In another aspect, the u-NGAL level isreduced by at least 4 fold relative to the u-NGAL level of a patienttransfused with conventionally stored blood. In another aspect, theu-NGAL level is reduced by at least 5 fold relative to the u-NGAL levelof a patient transfused with conventionally stored blood. In a furtheraspect, the u-NGAL level is reduced by at least 6 fold relative to theu-NGAL level of a patient transfused with conventionally stored blood.In another aspect, the u-NGAL level is reduced by at least 7 foldrelative to the u-NGAL level of a patient transfused with conventionallystored blood. In another aspect, the u-NGAL level is reduced by at least8 fold relative to the u-NGAL level of a patient transfused withconventionally stored blood. In another aspect, the u-NGAL level isreduced by at least 9 fold. In another aspect, the u-NGAL level isreduced by at least 10 fold. In a further aspect, the u-NGAL level isreduced by at least 50 fold.

Methods of the present disclosure provide for, and include, reducingserum creatinine levels in a trauma patient in need of transfusiontherapy comprising providing a trauma patient with oxygen reduced storedblood that has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the serum creatinine level is reduced by at least5% relative to the serum creatinine level of a patient transfused withconventionally stored blood. In another aspect, the serum creatininelevel is reduced by at least 10% relative to the serum creatinine levelof a patient transfused with conventionally stored blood. In anotheraspect, the serum creatinine level is reduced by at least 20% relativeto the serum creatinine level of a patient transfused withconventionally stored blood. In another aspect, the serum creatininelevel is reduced by at least 30% relative to the serum creatinine levelof a patient transfused with conventionally stored blood. In anotheraspect, the serum creatinine level is reduced by at least 40% relativeto the serum creatinine level of a patient transfused withconventionally stored blood. In another aspect, the serum creatininelevel is reduced by at least 50% relative to the serum creatinine levelof a patient transfused with conventionally stored blood. In anotheraspect, the serum creatinine level is reduced by at least 60% relativeto the serum creatinine level of a patient transfused withconventionally stored blood. In another aspect, the serum creatininelevel is reduced by at least 70% relative to the serum creatinine levelof a patient transfused with conventionally stored blood. In yet anotheraspect, the serum creatinine level is reduced by at least 80% relativeto the serum creatinine level of a patient transfused withconventionally stored blood. In a further aspect, the serum creatininelevel is reduced by at least 90% relative to the serum creatinine levelof a patient transfused with conventionally stored blood. In a furtheraspect, the serum creatinine level is reduced by between 1 and 10%, 10and 20%, 20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70and 80%, 80 and 90%, or 90 and 95% relative to the serum creatininelevel of a patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducingserum creatinine levels in a trauma patient in need of transfusiontherapy by between 1.5 and 10 fold comprising providing a trauma patientwith oxygen reduced stored blood that has an oxygen saturation of 20% orless prior to and during storage. In an aspect, the serum creatininelevel is reduced by between 2 and 3 fold relative to the serumcreatinine level of a patient transfused with conventionally storedblood. In another aspect, the serum creatinine level is reduced bybetween 3 and 4 fold relative to the serum creatinine level of a patienttransfused with conventionally stored blood. In another aspect, theserum creatinine level is reduced by between 4 and 10 fold relative tothe serum creatinine level of a patient transfused with conventionallystored blood. In another aspect, the serum creatinine level is reducedby between 6 and 9 fold relative to the serum creatinine level of apatient transfused with conventionally stored blood. In a furtheraspect, the serum creatinine level is reduced by between 2 and 5 foldrelative to the serum creatinine level of a patient transfused withconventionally stored blood. In another aspect, the serum creatininelevel is reduced by between 10 and 50 fold relative to the serumcreatinine level of a patient transfused with conventionally storedblood.

Methods of the present disclosure provide for, and include, reducingserum creatinine levels in a trauma patient in need of transfusiontherapy by at least 1.5 fold comprising providing a trauma patient withoxygen reduced stored blood that has an oxygen saturation of 20% or lessprior to and during storage. In an aspect, the serum creatinine level isreduced by at least 2 fold relative to the serum creatinine level of apatient transfused with conventionally stored blood. In another aspect,the serum creatinine level is reduced by at least 3 fold relative to theAST level of a patient transfused with conventionally stored blood. Inanother aspect, the serum creatinine level is reduced by at least 4 foldrelative to the serum creatinine level of a patient transfused withconventionally stored blood. In another aspect, the serum creatininelevel is reduced by at least 5 fold. In a further aspect, the serumcreatinine level is reduced by at least 6 fold relative to the serumcreatinine level of a patient transfused with conventionally storedblood. In another aspect, the serum creatinine level is reduced by atleast 7 fold relative to the serum creatinine level of a patienttransfused with conventionally stored blood. In another aspect, theserum creatinine level is reduced by at least 8 fold relative to theserum creatinine level of a patient transfused with conventionallystored blood. In another aspect, the serum creatinine level is reducedby at least 9 fold relative to the serum creatinine level of a patienttransfused with conventionally stored blood. In another aspect, theserum creatinine level is reduced by at least 10 fold relative to theserum creatinine level of a patient transfused with conventionallystored blood.

Methods of the present disclosure provide for, and include, reducingserum creatinine levels in a trauma patient in need of transfusiontherapy to between 0.5 and 1.5 mg/dL comprising providing a traumapatient with oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage. In an aspect, the serumcreatinine level is reduced to between 0.5 and 1 mg/dL relative to theserum creatinine level of a patient transfused with conventionallystored blood. In an aspect, the serum creatinine level is reduced tobetween 0.8 and 1 mg/dL relative to the serum creatinine level of apatient transfused with conventionally stored blood. In another aspect,the serum creatinine level is reduced to between 0.7 and 1.5 mg/dLrelative to the serum creatinine level of a patient transfused withconventionally stored blood.

Methods of the present disclosure provide for, and include, reducingserum creatinine levels in a trauma patient in need of transfusiontherapy to less than 1.5 mg/dL comprising providing a trauma patientwith oxygen reduced stored blood that has an oxygen saturation of 20% orless prior to and during storage. In an aspect, the serum creatininelevel is reduced to less than 1.4 mg/dL relative to the serum creatininelevel of a patient transfused with conventionally stored blood. In anaspect, the serum creatinine level is reduced to less than 1 mg/dL. Inanother aspect, the serum creatinine level is reduced to less than 0.8mg/dL relative to the serum creatinine level of a patient transfusedwith conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing BUNlevels in a trauma patient comprising providing a trauma patient in needof transfusion therapy with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. In anaspect, the BUN level is reduced by at least 5% relative to the BUNlevel of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 10% relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 20% relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 30% relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 40% relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 50% relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 60% relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 70% relative to theBUN level of a patient transfused with conventionally stored blood. Inyet another aspect, the BUN level is reduced by at least 80% relative tothe BUN level of a patient transfused with conventionally stored blood.In a further aspect, the BUN level is reduced by at least 90% relativeto the BUN level of a patient transfused with conventionally storedblood. In a further aspect, the BUN level is reduced by between 1 and10%, 10 and 20%, 20 and 30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and70%, 70 and 80%, 80 and 90%, or 90 and 95% relative to the BUN level ofa patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing BUNlevels in a trauma patient by between 1.5 and 10 fold comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. In anaspect, the BUN level is reduced by between 2 and 3 fold relative to theBUN level of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by between 3 and 4 folds. Inanother aspect, the BUN level is reduced by between 4 and 10 foldrelative to the BUN level of a patient transfused with conventionallystored blood. In another aspect, the BUN level is reduced by between 6and 9 fold relative to the BUN level of a patient transfused withconventionally stored blood. In a further aspect, the BUN level isreduced by between 2 and 5 fold relative to the BUN level of a patienttransfused with conventionally stored blood. In another aspect, the BUNlevel is reduced by between 10 and 100 fold relative to the BUN level ofa patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing BUNlevels in a trauma patient by at least 1.5 fold comprising providing atrauma patient in need of transfusion therapy with oxygen reduced storedblood that has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the BUN level is reduced by at least 2 foldrelative to the BUN level of a patient transfused with conventionallystored blood. In another aspect, the BUN level is reduced by at least 3fold relative to the BUN level of a patient transfused withconventionally stored blood. In another aspect, the BUN level is reducedby at least 4 fold relative to the BUN level of a patient transfusedwith conventionally stored blood. In another aspect, the BUN level isreduced by at least 5 fold relative to the BUN level of a patienttransfused with conventionally stored blood. In a further aspect, theBUN level is reduced by at least 6 fold. In another aspect, the BUNlevel is reduced by at least 7 fold relative to the BUN level of apatient transfused with conventionally stored blood. In another aspect,the BUN level is reduced by at least 8 fold relative to the BUN level ofa patient transfused with conventionally stored blood. In anotheraspect, the BUN level is reduced by at least 9 fold relative to the BUNlevel of a patient transfused with conventionally stored blood. Inanother aspect, the BUN level is reduced by at least 10 fold relative tothe BUN level of a patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing thepercentage of CD45+ neutrophils in a trauma patient comprising providinga trauma patient with oxygen reduced stored blood that has an oxygensaturation of 20% or less prior to and during storage. In an aspect, thepercentage of CD45+ neutrophils is reduced by at least 5% relative tothe CD45+ neutrophil level of a patient transfused with conventionallystored blood. In another aspect, the percentage of CD45+ neutrophils isreduced by at least 10% relative to the CD45+ neutrophil level of apatient transfused with conventionally stored blood. In another aspect,the percentage of CD45+ neutrophils is reduced by at least 20% relativeto the CD45+ neutrophil level of a patient transfused withconventionally stored blood. In another aspect, the percentage of CD45+neutrophils is reduced by at least 30% relative to the CD45+ neutrophillevel of a patient transfused with conventionally stored blood. Inanother aspect, the percentage of CD45+ neutrophils is reduced by atleast 40% relative to the CD45+ neutrophil level of a patient transfusedwith conventionally stored blood. In another aspect, the percentage ofCD45+ neutrophils is reduced by at least 50% relative to the CD45+neutrophil level of a patient transfused with conventionally storedblood. In another aspect, the percentage of CD45+ neutrophils is reducedby at least 60% relative to the CD45+ neutrophil level of a patienttransfused with conventionally stored blood. In another aspect, thepercentage of CD45+ neutrophils is reduced by at least 70% relative tothe CD45+ neutrophil level of a patient transfused with conventionallystored blood. In yet another aspect, the percentage of CD45+ neutrophilsis reduced by at least 80% relative to the CD45+ neutrophil level of apatient transfused with conventionally stored blood. In a furtheraspect, the percentage of CD45+ neutrophils is reduced by at least 90%relative to the CD45+ neutrophil level of a patient transfused withconventionally stored blood. In a further aspect, the percentage ofCD45+ neutrophils is reduced by between 1 and 10%, 10 and 20%, 20 and30%, 30 and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70 and 80%, 80 and90%, or 90 and 95% relative to the CD45+ neutrophil level of a patienttransfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing thepercentage of CD45+ neutrophils in a trauma patient in need oftransfusion therapy by between 1.5 and 10 fold comprising providing atrauma patient with oxygen reduced stored blood that has an oxygensaturation of 20% or less prior to and during storage. In an aspect, thepercentage of CD45+ neutrophils is reduced by between 2 and 3 foldrelative to the CD45+ neutrophil level of a patient transfused withconventionally stored blood. In another aspect, the percentage of CD45+neutrophils is reduced by between 3 and 4 fold relative to the CD45+neutrophil level of a patient transfused with conventionally storedblood. In another aspect, the percentage of CD45+ neutrophils is reducedby between 4 and 10 fold relative to the CD45+ neutrophil level of apatient transfused with conventionally stored blood. In another aspect,the percentage of CD45+ neutrophils is reduced by between 6 and 9 foldrelative to the CD45+ neutrophil level of a patient transfused withconventionally stored blood. In a further aspect, the percentage ofCD45+ neutrophils is reduced by between 2 and 5 fold relative to theCD45+ neutrophil level of a patient transfused with conventionallystored blood. In another aspect, the percentage of CD45+ neutrophils isreduced by between 10 and 50 fold relative to the CD45+ neutrophil levelof a patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing thepercentage of CD45+ neutrophils in a trauma patient in need oftransfusion therapy by at least 1.5 fold comprising providing a traumapatient with oxygen reduced stored blood that has an oxygen saturationof 20% or less prior to and during storage. In an aspect, the percentageof CD45+ neutrophils is reduced by at least 2 fold relative to the CD45+neutrophil level of a patient transfused with conventionally storedblood. In another aspect, the percentage of CD45+ neutrophils is reducedby at least 3 fold relative to the CD45+ neutrophil level of a patienttransfused with conventionally stored blood. In another aspect, thepercentage of CD45+ neutrophils is reduced by at least 4 fold relativeto the CD45+ neutrophil level of a patient transfused withconventionally stored blood. In another aspect, the percentage of CD45+neutrophils is reduced by at least 5 fold relative to the CD45+neutrophil level of a patient transfused with conventionally storedblood. In a further aspect, the percentage of CD45+ neutrophils isreduced by at least 6 fold relative to the CD45+ neutrophil level of apatient transfused with conventionally stored blood. In another aspect,the percentage of CD45+ neutrophils is reduced by at least 7 foldrelative to the CD45+ neutrophil level of a patient transfused withconventionally stored blood. In another aspect, the percentage of CD45+neutrophils is reduced by at least 8 fold relative to the CD45+neutrophil level of a patient transfused with conventionally storedblood. In another aspect, the percentage of CD45+ neutrophils is reducedby at least 9 fold relative to the CD45+ neutrophil level of a patienttransfused with conventionally stored blood. In another aspect, thepercentage of CD45+ neutrophils is reduced by at least 10 fold relativeto the CD45+ neutrophil level of a patient transfused withconventionally stored blood.

Methods of the present disclosure provide for, and include, reducing theCXCL1 levels in a trauma patient in need of transfusion therapycomprising providing a trauma patient with oxygen reduced stored bloodthat has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the CXCL1 level is reduced by at least 5%relative to the CXCL1 level of a patient transfused with conventionallystored blood. In another aspect, the CXCL1 level is reduced by at least10% relative to the CXCL1 level of a patient transfused withconventionally stored blood. In another aspect, the CXCL1 level isreduced by at least 20% relative to the CXCL1 level of a patienttransfused with conventionally stored blood. In another aspect, theCXCL1 level is reduced by at least 30% relative to the CXCL1 level of apatient transfused with conventionally stored blood. In another aspect,the CXCL1 level is reduced by at least 40% relative to the CXCL1 levelof a patient transfused with conventionally stored blood. In anotheraspect, the CXCL1 level is reduced by at least 50% relative to the CXCL1level of a patient transfused with conventionally stored blood. Inanother aspect, the CXCL1 level is reduced by at least 60% relative tothe CXCL1 level of a patient transfused with conventionally storedblood. In another aspect, the CXCL1 level is reduced by at least 70%relative to the CXCL1 level of a patient transfused with conventionallystored blood. In yet another aspect, the CXCL1 level is reduced by atleast 80% relative to the CXCL1 level of a patient transfused withconventionally stored blood. In a further aspect, the CXCL1 level isreduced by at least 90% relative to the CXCL1 level of a patienttransfused with conventionally stored blood. In a further aspect, theCXCL1 level is reduced by between 1 and 10%, 10 and 20%, 20 and 30%, 30and 40%, 40 and 50%, 50 and 60%, 60 and 70%, 70 and 80%, 80 and 90%, or90 and 95% relative to the CXCL1 level of a patient transfused withconventionally stored blood

Methods of the present disclosure provide for, and include, reducing theCXCL1 levels in a trauma patient by between 1.5 and 10 fold comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and 30 during storage. In anaspect, the CXCL1 level is reduced by between 2 and 3 fold relative tothe CXCL1 level of a patient transfused with conventionally storedblood. In another aspect, the CXCL1 level is reduced by between 3 and 4fold relative to the CXCL1 level of a patient transfused withconventionally stored blood. In another aspect, the CXCL1 level isreduced by between 4 and 10 fold relative to the CXCL1 level of apatient transfused with conventionally stored blood. In another aspect,the CXCL1 level is reduced by between 6 and 9 fold relative to the CXCL1level of a patient transfused with conventionally stored blood. In afurther aspect, the CXCL1 level is reduced by between 2 and 5 foldrelative to the CXCL1 level of a patient transfused with conventionallystored blood. In another aspect, the CXCL1 level is reduced by between10 and 100 fold relative to the CXCL1 level of a patient transfused withconventionally stored blood.

Methods of the present disclosure provide for, and include, reducing theCXCL1 levels in a trauma patient by at least 1.5 fold comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage.

In an aspect, the CXCL1 level is reduced by at least 2 fold relative tothe CXCL1 level of a patient transfused with conventionally storedblood. In another aspect, the CXCL1 level is reduced by at least 3 foldrelative to the CXCL1 level of a patient transfused with conventionallystored blood. In another aspect, the CXCL1 level is reduced by at least4 fold relative to the CXCL1 level of a patient transfused withconventionally stored blood. In another aspect, the CXCL1 level isreduced by at least 5 fold relative to the CXCL1 level of a patienttransfused with conventionally stored blood. In a further aspect, theCXCL1 level is reduced by at least 6 fold relative to the CXCL1 level ofa patient transfused with conventionally stored blood. In anotheraspect, the CXCL1 level is reduced by at least 7 fold relative to theCXCL1 level of a patient transfused with conventionally stored blood. Inanother aspect, the CXCL1 level is reduced by at least 8 fold relativeto the CXCL1 level of a patient transfused with conventionally storedblood. In another aspect, the CXCL1 level is reduced by at least 9 foldrelative to the CXCL1 level of a patient transfused with conventionallystored blood. In another aspect, the CXCL1 level is reduced by at least10 fold relative to the CXCL1 level of a patient transfused withconventionally stored blood.

Methods of the present disclosure provide for, and include, reducing theIL-6 levels in a trauma patient in need of transfusion therapycomprising providing a trauma patient with oxygen reduced stored bloodthat has an oxygen saturation of 20% or less prior to and duringstorage. In an aspect, the IL-6 level is reduced by at least 5% relativeto the IL-6 level of a patient transfused with conventionally storedblood. In another aspect, the IL-6 level is reduced by at least 10%relative to the IL-6 level of a patient transfused with conventionallystored blood. In another aspect, the IL-6 level is reduced by at least20%. In another aspect, the IL-6 level is reduced by at least 30%relative to the IL-6 level of a patient transfused with conventionallystored blood. In another aspect, the IL-6 level is reduced by at least40% relative to the IL-6 level of a patient transfused withconventionally stored blood. In another aspect, the IL-6 level isreduced by at least 50% relative to the IL-6 level of a patienttransfused with conventionally stored blood. In another aspect, the IL-6level is reduced by at least 60% relative to the IL-6 level of a patienttransfused with conventionally stored blood.

In another aspect, the IL-6 level is reduced by at least 70% relative tothe IL-6 level of a patient transfused with conventionally stored blood.In yet another aspect, the IL-6 level is reduced by at least 80%relative to the IL-6 level of a patient transfused with conventionallystored blood. In a further aspect, the IL-6 level is reduced by at least90% relative to the IL-6 level of a patient transfused withconventionally stored blood. In a further aspect, the IL-6 level isreduced by between 1 and 10%, 10 and 20%, 20 and 30%, 30 and 40%, 40 and50%, 50 and 60%, 60 and 70%, 70 and 80%, 80 and 90%, or 90 and 95%relative to the IL-6 level of a patient transfused with conventionallystored blood.

Methods of the present disclosure provide for, and include, reducing theIL-6 levels in a trauma patient by between 1.5 and 10 fold comprisingproviding a trauma patient with oxygen reduced stored blood that has anoxygen saturation of 20% or less prior to and during storage. In anaspect, the IL-6 level is reduced by between 2 and 3 fold relative tothe IL-6 level of a patient transfused with conventionally stored blood.In another aspect, the IL-6 level is reduced by between 3 and 4 foldrelative to the IL-6 level of a patient transfused with conventionallystored blood. In another aspect, the IL-6 level is reduced by between 4and 10 folds. In another aspect, the IL-6 level is reduced by between 6and 9 fold relative to the IL-6 level of a patient transfused withconventionally stored blood. In a further aspect, the IL-6 level isreduced by between 2 and 5 fold relative to the IL-6 level of a patienttransfused with conventionally stored blood. In another aspect, the IL-6level is reduced by between 10 and 100 fold relative to the IL-6 levelof a patient transfused with conventionally stored blood.

Methods of the present disclosure provide for, and include, reducing theIL-6 levels in a trauma patient by at least 1.5 fold comprisingproviding a trauma patient in need of transfusion therapy with oxygenreduced stored blood that has an oxygen saturation of 20% or less priorto and during storage. In an aspect, the IL-6 level is reduced by atleast 2 fold relative to the IL-6 level of a patient transfused withconventionally stored blood. In another aspect, the IL-6 level isreduced by at least 3 fold relative to the IL-6 level of a patienttransfused with conventionally stored blood. In another aspect, the IL-6level is reduced by at least 4 fold relative to the IL-6 level of apatient transfused with conventionally stored blood. In another aspect,the IL-6 level is reduced by at least 5 fold. In a further aspect, theIL-6 level is reduced by at least 6 fold relative to the IL-6 level of apatient transfused with conventionally stored blood. In another aspect,the IL-6 level is reduced by at least 7 fold relative to the IL-6 levelof a patient transfused with conventionally stored blood. In anotheraspect, the IL-6 level is reduced by at least 8 fold relative to theIL-6 level of a patient transfused with conventionally stored blood. Inanother aspect, the IL-6 level is reduced by at least 9 fold relative tothe IL-6 level of a patient transfused with conventionally stored blood.In another aspect, the IL-6 level is reduced by at least 10 foldrelative to the IL-6 level of a patient transfused with conventionallystored blood.

As used herein, the terms “higher”, “greater” or “increased” means thatthe measured values of oxygen reduced and anaerobically stored blood,when compared to the measured values of otherwise equivalently treatedconventionally stored blood, are at least 1 standard deviation greater,with a sample size of at least 2 for each compared measured condition.

As used herein, the terms “reduce”, “reduced”, “lower”, “decreased” or“less” means that the measured values of oxygen reduced andanaerobically stored blood when compared to the measured values ofotherwise equivalently treated normoxic or hyperoxic conventionallystored blood RBCs, are at least 1 standard deviation lower, with asample size of at least 5 for each compared measured condition.

As used herein the term “about” refers to±10%.

As used herein the term “less than” refers to a smaller amount and anamount greater than zero.

The terms “comprises,” “comprising,” “includes,” “including,” “having,”and their conjugates mean “including but not limited to.”

The term “consisting of” means “including and limited to.”

The term “consisting essentially of” means that the composition, methodor structure can include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof

As used herein, the term “blood” refers to whole blood, leukoreducedRBCs, platelet reduced RBCs, and leukocyte and platelet reduced RBCs.The term blood further includes packed red blood cells, platelet reducedpacked red blood cells, leukocyte reduced packed red blood cells, andleukocyte and platelet reduced packed red blood cells. The temperatureof blood can vary depending on the stage of the collection process,starting at the normal body temperature of 37° C. at the time and pointof collection, but decreasing rapidly to about 30° C. as soon as theblood leaves the patient's body and further thereafter to roomtemperature in about 6 hours when untreated, and ultimately beingrefrigerated at between about 4° C. and 6° C. Human red blood cells invivo are in a dynamic state. The red blood cells contain hemoglobin, theiron-containing protein that carries oxygen throughout the body andgives red blood its color. The percentage of blood volume composed ofred blood cells is called the hematocrit. As used herein, unlessotherwise limited, RBCs also includes packed red blood cells (pRBCs).Packed red blood cells are prepared from whole blood usingcentrifugation techniques commonly known in the art. As used herein,unless otherwise indicated, the hematocrit of pRBCs is about 70%. Asused herein, oxygen reduced stored RBCs can include oxygen and carbondioxide reduced stored RBCs. As used herein, oxygen reduced (OR) bloodcan include oxygen and carbon dioxide (OCR) reduced blood.

As used herein the terms “patient” and “subject” is used interchangeablyto mean a person or animal in need of transfusion.

As used herein the term “trauma” includes exsanguination, hemorrhagictrauma.

As used herein the term “hemorrhagic shock” is shock brought on by aloss of circulating blood volume and/or oxygen carrying capacity.Hemorrhagic shock results from any condition associated with blood loss,internal (e.g., gastrointestinal bleeding) or external hemorrhage, andtrauma (e.g., penetrating or blunt trauma), among others.

As used herein the term “adverse event” includes an event resulting fromhemorrhagic shock in a hemorrhagic trauma patient.

As used herein the terms “injury”, “damage”, and “failure” refer to anorgan not functioning properly or not functioning as is expected in aperson or animal without disease or injury.

As used herein, a “unit” of blood is about 450-500 ml includinganticoagulant. Suitable anticoagulants include CPD, CPDA1, ACD, andACD-A.

Throughout this application, various aspects of this disclosure may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as “from 1 to 6” should be considered to havespecifically disclosed subranges such as “from 1 to 3,” “from 1 to 4,”“from 1 to 5,” “from 2 to 4,” “from 2 to 6,” “from 3 to 6,” etc., aswell as individual numbers within that range, for example, 1, 2, 3, 4,5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

As used herein the term “method” refers to manners, means, techniques,and procedures for accomplishing a given task including, but not limitedto, providing a human patient in need of a blood transfusion with oxygenreduced stored blood having an initial oxygen saturation of 20% or lessand stored for at least 2 days.

While the present disclosure has been described with reference toparticular embodiments, it will be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the scope of the presentdisclosure. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the presentdisclosure without departing from the scope of the present disclosure.

Therefore, it is intended that the present disclosure not be limited tothe particular embodiments disclosed as the best mode contemplated forcarrying out the present disclosure, but that the present disclosurewill include all embodiments falling within the scope and spirit of theappended claims.

EXAMPLES Example 1 Collection of Blood and Sample Preparation

Each pool of red blood cells are collected from a total of 12-14 rats inCP2D anticoagulant. The pooled blood is leukoreduced using neonatalleukoreduction filter, component separated and RBCs are stored in AS-3additive solution. Total of two pools of RBCs are collected. Each poolis split four ways: Unprocessed control (C), sham control (SC),oxygen-reduced (OR) and oxygen and carbon dioxide reduced (OCR). For C,SC, OR and OCR units, RBC subunit is processed by transferring into 80mL PVC blood transfer bag and final RBC products are made by gasexchange process. The RBC bags except for C are filled with 100% N₂ (forOR), or 95% N₂/5% CO₂ (for OCR) or air (SC) through sterile filter andgently rotated on its long side at 2-3 RPM (except for C). For OR andOCR units, after 10 minutes, gas is removed through the filter and freshgas is introduced for subsequent gas exchange process. This process isrepeated 5-8 times until target %SO2 of 5-10% as measured by ABL-90cooximeter (Radiometer Copenhagen) is achieved. SC unit is rotated 15without any gas exchange for 60 minutes. OR and OCR units are storedanaerobically in a N₂-filled canister, while C and SC units are storedin ambient air. All units are stored for 3 weeks at 4° C. and sampled atdays 0 or 1, 7, 14, 21, and 28. Two pools were prepared and stored.

On days 0, 1, 7, 14, 21, and 28, ATP, 2,3-DPG, and hemolysis analysisare performed. As shown in FIG. 1, ATP levels are higher in OR-blood atday 21 and OCR-blood at days 7, 14, 21, and 28 compared toconventionally stored blood (control). OR-blood also has higher levelsof 2.3-DPG at days 2, 7, and 14, compared to control. OCR-blood alsoshows a higher level of 2,3-DPG on days 2, 7, and 14 compared tocontrol. See FIG. 2.

Example 2 Recovery of Oxygen Reduced Blood

A small volume (less than 200μL) of Control, OR-, and OCR-blood storedfor 3 weeks is labeled with techniteum-99 m. Animals are transfused withlabeled RBC (less than 200 uL) and circulating radioactivity is measuredperiodically up to 24 hours in order to estimate fraction of transfusedRBC surviving 24 hours after transfusion. As shown in FIG. 3,significantly more OR- and OCR-RBCs are recovered compared to controlRBCs when RBCs were stored for three weeks.

Example 3 Rat Model of Hemorrhagic Shock Resuscitation

Collection of blood and sample preparation: Each pool of red blood cellsare collected from a total of 12-14 rats in CP2D anticoagulant. Thepooled blood is leukoreduced using neonatal leukoreduction filter,component separated and RBCs are stored in AS-3 additive solution. Totalof six pools of RBCs are collected. Two pools are prepared forconventional storage (control). Two pools are depleted of oxygen (oxygenreduced; OR), and the remaining two pools of blood are depleted ofoxygen and carbon dioxide (oxygen and carbon dioxide reduced; OCR). Eachof the four pools to be reduced is processed by transferring RBCs into600 mL PVC blood transfer bag and final RBC products are made by gasexchange process. The RBC bag is filled with 100% N₂ (for OR), or 95%N₂/5% CO₂ (for OCR) through sterile filter and gently rotated on itslong side at 60-90 RPM. After 10 minutes, gas is removed through thefilter and fresh gas is introduced for subsequent gas exchange process.This process is repeated 5-8 times until target %SO2 of 5-10% asmeasured by ABL-90 cooximeter (Radiometer Copenhagen) is achieved. ORand OCR blood is stored anaerobically in a N₂-filled canister.

Studies are performed in Sprague-Dawley rats (Charles RiverLaboratories, Boston, Mass.) weighing 150-200 grams (g). Briefly,animals are anesthetized by administering 40 mg/kg of sodiumpentobarbital intraperitoneally. Animals are placed in the supineposition on a heating pad to maintain core body temperature at 37° C.Animals are prepared with: (i) a left jugular vein and left femoralartery catheterization, (ii) tracheotomy (polyethylene-90 tube), and(iii) left ventricle (LV) conductance catheter introduction through theright carotid artery. Animals are mechanically ventilated (TOPOventilator, Kent Scientific, Torrington, Conn.) using room air, with arespiration rate of 50-70 breaths per min and a peak inspiratorypressure of 10-15 cmH₂O. After instrumentation, volatile anesthesia(1.5%/vol Isoflurane, Dragerwerk AG, Laubeck, Germany) is administeredusing a vaporizer connected to the ventilator. Depth of anesthesia iscontinually verified via toe pinch, as needed, isoflurane was increasedby 0.1%/vol to prevent animal discomfort.

Anesthetized animals are hemorrhaged by withdrawing 50% of the animal'sblood volume (BV; estimated 7% of body weight) via the femoral arterycatheter within 10 min, placing the animals in a hypovolemic shockcondition. The hypovolemic shock condition is maintained for 30 min.Resuscitation is implemented by infusion of previously stored RBCs at300 microliters per min (μL/min) via the femoral artery until Meanarterial pressure (MAP) is stabilized at 90% of the baseline during 60minutes resuscitation period. At 10, 20, 30, 45 and 60 minutes duringthis period, MAP and heart rate (HR) are obtained from a femoral arterycatheter (PowerLab, AD Instruments, Colorado Springs, Colo.). After 60mins, hematocrit (Hct) is measured via centrifugation of heparinizedcapillary tubes. Hemoglobin (Hb), lactate, glucose, K+, Na+, pH,arterial blood gas are determined by ABL90 cooximeter (Radiometer,Copenhagen). Indices of cardiac function and systemic values (MAP, HR,Hct, Hb, and blood gases) are monitored at baseline (BL), during shock,and 10 (Early R), 20, 30, 45, and 60 (Late R) mins post resuscitation.Animals are euthanized at the end of the experiment.

Example 4 Hematocrit Analysis in a Rat Model of Hemorrhagic Shock

Hematocrit (Hct) is reduced by approximately 30 to 40% after hypovolemicshock is induced. Providing conventionally, OR, or OCR blood stored for1 week is capable of restoring hematocrit to normal levels. See FIG. 4A.However, as shown in FIG. 4B, OR-blood stored for one week show anincreased percent hematocrit compared to control and OCR-blood after 10mins of resuscitation (Early R). The percent hematocrit of OR-bloodremains improved compared to control after 60 mins (Late R) ofresuscitation.

Example 5 Mean Arterial Pressure Changes with Oxygen Reduced Blood

Mean arterial pressure (MAP) is obtained from the femoral arterycatheter (PowerLab, AD Instruments, Colorado Springs, Colo.). As shownin FIG. SA, baseline MAP is between 80 and 110 mmHg. MAP is reduced tobetween 20 and 60 mmHg during

hemorrhagic shock. Resuscitation of animals with OR and OCR blood storedfor one week increases the MAP to approximately 80 and 90 mmHg,respectively. As shown in FIG. 5B, resuscitation with OR blood, after 10mins, is able to restore MAP to normal range compared to control.Control and OCR stored blood is able to restore MAP to a normal rangeafter 60 mins of resuscitation. The amount of blood required toresuscitate and preserve hemodynamics with conventionally stored RBCs(control) was greater than OR and OCR RBCs required. See FIG. 6A andFIG. 6B.

Example 6 Metabolic Reaction to Hemorrhagic Shock

Hemorrhagic shock in animals increases the lactate level from about 2mmol/L to between about 8 and 14 mmol/L. Resuscitation with OR and OCRRBCs stored for one week reduces lactate levels to near normal levelsafter just 10 mins of resuscitation. See FIG. 7A Lactate levels ofanimals resuscitated with control blood are similar to lactate levels ofanimals in hemorrhagic shock. Animals treated with control, OR and OCRRBCs for 60 mins show similar lactate levels. As shown, in FIG. 7B, ORRBCs stored for 3 weeks are also able to reduce lactate levels comparedto control after 10 mins of resuscitation. However, after 80 mins ofresuscitation OCR RBCs restored lactate levels to a normal range.Control and OR RBCs were able to reduce lactate levels but not to thenormal range of 1 to 3 mmol/L. Analysis of glucose levels show that thenormal range of about 160 mg/dL to about 240 mg/dL glucose is increasedto a range of about 320 to about 510 mg/dL in animals under hemorrhagicshock. See .FIG. 8A and FIG. 8B. Both OR and OCR RBCs stored for oneweek decrease glucose levels compared to control after 10 mins ofresuscitation. All three samples restored glucose levels to the normalrange after 60 mins of resuscitation. As shown in FIG. 8B, OR and OCRRBCs stored for three weeks are also able to decrease glucose levelscompared to control after 10 mins of resuscitation. Unlike the RBCsstored for one week, only OR and OCR RBCs were able to restore glucosewithin the normal range. Thus, both lactate and glucose levels arereduced faster in OR and OCR RBCs compared to control RBCs.

Example 7 Vital Organ Injury and Inflammation

Animals are analyzed for organ injury and inflammation afterexperiencing hemorrhagic shock and resuscitation. Elevated levels ofliver enzymes signify some form of liver damage or injury. Aspartateaminotransferase (AST) and alanine aminotransferase (ALT) levels wereanalyzed to determine liver damage. Resuscitation with OR and OCR RBCspreviously stored for one week (FIG. 9A and FIG. 10A) and three weeks(FIG. 9B and FIG. 10B) reduced AST and ALT levels compared control RBCsstored for the same period of time. Serum creatinine and blood ureanitrogen (BUN) levels were analyzed to determine kidney function. OR andOCR RBCs stored for one week reduced serum creatinine levels greaterthan 30% compared to control RBCs (FIG. 11A). After three weeks ofstorage serum creatinine levels of animals treated with control, OR, andOCR RBCs overlap (FIG. 11B). BUN levels are decreased by greater than30% in animals treated with OCR RBCs stored for one week compared tocontrol (FIG. 12A). BUN levels also decreased by greater than 30% inanimals treated with OR RBCs stored for three weeks compared to control(FIG. 12B). Overall, vital organ function was preserved with OR and OCRRBCs compared to control RBCs

Liver, spleen and lungs are resected from animals upon completion of thein vivo studies and analyzed for various inflammatory factors includingCXCL1 , urinary neutrophil gelatinase-associated lipocalin (u-NGAL),IL-6, and neutrophils. CXCL1 is reduced in animals treated with OR andOCR RBCs stored for one or three weeks, compared to control stored forthe same period of time (FIG. 13A,B, FIG. 14A, B, and FIG. 15A, B). Asshown in FIGS. 16A and B, u-NGAL is reduced in the kidneys of animalstreated with OR or OCR RBCs, stored for one or three weeks, compared tocontrol RBCs stored for an equivalent amount of time (FIG. 16). As shownin FIGS. 17 and 18, the percentage of lungs resected from animals withCD45+ neutrophils and the level of IL-6 is significantly decreased in ORand OCR RBCs compared to control RBCs stored for the same period oftime. These results show that organ injury and inflammation is decreasedin animals treated with OR and OCR RBCs compared to animals treated withcontrol RBCs.

1. A method for treating low mean arterial pressure in a subject in needthereof comprising providing stored oxygen reduced blood to a subjecthaving a low mean arterial pressure resulting from a hemorrhagic trauma,said oxygen reduced blood having an initial oxygen saturation of 20% orless and maintained at an oxygen saturation of 20% or less for a storageperiod.
 2. The method of claim 1, wherein said mean arterial pressure insaid subject in need thereof is increased relative to a patientreceiving conventionally stored blood after said providing.
 3. Themethod of claim 1, wherein said hemorrhagic trauma is selected from thegroup consisting of surgery, a penetrating wound, blunt force trauma,injury due to a fall, and injury due to a car accident.
 4. The method ofclaim 1, wherein said increasing mean arterial pressure in said subjectin need thereof is at a rate faster than the rate of increase of meanarterial pressure in a subject not receiving said stored oxygen reducedblood.
 5. The method of claim 1, wherein said mean arterial pressure insaid subject in need thereof is increased by at least 20% after saidproviding, relative to the mean arterial pressure of a patienttransfused with conventionally stored blood.
 6. The method of claim 5,wherein said mean arterial pressure in said subject remains at saidincreased by at least 20% for at least 1 hour after said providing.
 7. Amethod for reducing the amount of blood needed for transfusion in atrauma patient in need thereof comprising providing oxygen reduced bloodhaving an oxygen saturation of 20% or less prior to and during storage.8. A method for reducing hemorrhagic shock in a trauma patient in needthereof comprising providing oxygen reduced blood having an oxygensaturation of 20% or less prior to and during storage, wherein saidtrauma patient comprises a lactate level of between 0.5 and 2.5 mmol/Lprior to said providing, and wherein said hemorrhagic shock is reversed.9. A method of reducing a liver injury in a trauma patient in need oftransfusion therapy comprising providing oxygen reduced blood having anoxygen saturation of 20% or less prior to and during storage.
 10. Amethod of claim 9, wherein said trauma patient has improved AST levels,ALT levels, or a combination thereof after said providing.
 11. A methodof reducing a kidney failure in a trauma patient in need of transfusiontherapy comprising providing oxygen reduced blood having an oxygensaturation of 20% or less prior to and during storage.
 12. The method ofclaim 11, wherein said trauma patient has improved levels selected fromneutrophil gelatinase-associated lipocalin (NGAL), serum creatinine,blood urea nitrogen (BUN), or a combination thereof.
 13. A method ofreducing a lung injury in a trauma patient in need of transfusiontherapy comprising providing oxygen reduced blood having an oxygensaturation of 20% or less prior to and during storage.
 14. A method ofreducing lactate in a trauma patient in need thereof comprisingproviding oxygen reduced blood having an oxygen saturation of 20% orless prior to and during storage.
 15. A method of reducing AST in atrauma patient in need thereof comprising providing oxygen reduced bloodhaving an oxygen saturation of 20% or less prior to and during storage.16. A method of reducing ALT in a trauma patient in need thereofcomprising providing oxygen reduced blood having an oxygen saturation of20% or less prior to and during storage.
 17. A method of reducing BUN ina trauma patient in need thereof comprising providing oxygen reducedblood having an oxygen saturation of 20% or less prior to and duringstorage.
 18. A method of reducing neutrophil gelatinase-associatedlipocalin (NGAL) in a trauma patient in need thereof comprisingproviding oxygen reduced blood having an oxygen saturation of 20% orless prior to and during storage.
 19. A method of reducing serumcreatinine in a trauma patient in need thereof comprising providingoxygen reduced blood having an oxygen saturation of 20% or less prior toand during storage.
 20. Use of donor blood to manufacture oxygen reducedblood having an oxygen saturation of 20% or less prior to and duringstorage for the therapeutic application of low mean arterial pressure.21. Use of donor blood to manufacture oxygen reduced blood having anoxygen saturation of 20% or less prior to and during storage for thetherapeutic application of reducing an adverse event in a hemorrhagictrauma patient, wherein said adverse event is selected from the groupcomprising liver injury, lung failure, kidney failure, and heartfailure.